Immobilized biologically active entities having a high degree of biological activity

ABSTRACT

The present invention relates to immobilized biologically active entities having heparin cofactor II binding activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application a continuation of application Ser. No. 13/208,220,filed Aug. 11, 2011, which is a divisional of Ser. No. 11/938,162, filedNov. 9, 2007, which is a continuation-in-part of co-pending applicationSer. No. 11/747,162, filed May 10, 2007, which is a continuation-in-partof co-pending application Ser. No. 11/433,105, filed May 12, 2006.

BACKGROUND OF THE INVENTION

In the field of medical devices, glass, polymeric, and/or metallicmaterials are common substrate materials. These materials can be usedfor diagnostic devices or extracorporeal devices. With the exception ofglass, many of the materials can be used for implantable devices.

Immobilization of biologically active entities on substrate materials ina biologically active form involves an appreciation of the respectivechemistries of the entity and the substrate material. Modification ofthe chemical composition of a substrate material may be required toimmobilize a biologically active entity thereon. This is usuallyaccomplished by treating surfaces of the substrate material to generatea population of chemically reactive elements or groups, followed byimmobilization of the biologically active entity with an appropriateprotocol. With other substrate materials, surfaces of a substratematerial are covered, or coated, with a material having reactivechemical groups incorporated therein. Biologically active entities arethen immobilized on the substrate material through the reactive chemicalgroups of the covering material. A variety of schemes for covering, orcoating, substrate materials have been described. Representativeexamples of biologically active entities immobilized to a substratematerial with a covering, or coating, material are described in U.S.Pat. Nos. 4,810,784; 5,213,898; 5,897,955; 5,914,182; 5,916,585; and6,461,665.

When biologically active compounds, compositions, or entities areimmobilized, the biological activity of these “biologics” can benegatively impacted by the process of immobilization. The biologicalactivity of many of biologics is dependent on the conformation (i.e.,primary, secondary, tertiary, etc.) of the biologic in its immobilizedstate. In addition to a carefully selected immobilization process,chemical alterations to the biologic may be required for the biologic tobe incorporated into the covering material with a conformation thatrenders the biologic sufficiently active to perform its intendedfunction.

Despite an optimized covering and immobilization scheme, the biologicalactivity of the immobilized biologic can be less than desired,particularly if additional processing, such as sterilization, isincluded. For implantable medical devices, sterilization is requiredprior to use. Sterilization may also be required for in vitro diagnosticdevices having sensitivity to contaminants. Sterilization of suchdevices usually requires exposure of the devices to elevatedtemperature, pressure, and humidity, often for several cycles. In someinstances, antibiotic agents, such as ethylene oxide gas (EtO) or vaporhydrogen peroxide are included in the sterilization process. In additionto sterilization, mechanical compaction and expansion, or long-termstorage of an immobilized biologic can degrade the activity of thebiologic.

There exists a need for medical devices having biologically activeentities immobilized thereon without significant loss of biologicalactivity, particularly when the immobilized biologically active entitiesare subjected to sterilization, mechanical compaction and expansion,and/or storage. Such a medical device would have biologically compatiblecompositions or compounds included with the immobilized biologicallyactive entities that serve to minimize degradation of the biologicalactivity of the entities during immobilization, sterilization,mechanical compaction and expansion, and/or storage. In some instances,the additional biologically compatible compositions or compounds wouldincrease the biological activity of some biologically active entitiesfollowing a sterilization procedure. Biologically active entities ofparticular interest for immobilization have anti-thrombotic properties.

SUMMARY OF THE INVENTION

The present invention relates to medical devices having substratematerials with biologically active entities having heparin cofactor IIbinding activity immobilized thereon. In some embodiments, thebiologically active entities are immobilized in combination withadditional biologically compatible organic chemical compositions thatenable the biologically active entities to retain significant heparincofactor II binding activity, especially following exposure of theimmobilized entities to processing and storage conditions that wouldotherwise degrade the biological activity of the entities. In someembodiments, the additional biologically compatible organic chemicalcompositions provide adjunctive functions to substrates or coatings towhich the biologically active entities are immobilized.

A suitable substrate material can be any material with a surface havingreactive chemical groups that are capable of attaching, confining, orotherwise immobilizing a biologically active entity in a biologicallyactive form to one or more surfaces of the substrate material. Substratematerials can also have a multiplicity of reactive chemical groups addedto surfaces of the materials through the application of one or morecovering compositions, or materials, to the surfaces. At least a portionof a covering material has chemical elements, groups, compounds, orcomponents that are reactive to biologically active entities and serveto attach, confine, or otherwise immobilize a biologically active entityin a biologically active form to the covering material. In someembodiments, the biologically active entity can be reversiblyimmobilized.

At least one type of biologically active entity is chemically attached,confined, or otherwise immobilized to suitable reactive chemical groupson the substrate material and/or covering material. Followingimmobilization of a plurality of biologically active entities to atleast a portion of a multiplicity of reactive chemical groups present ona substrate material and/or covering material, an additionalbiologically compatible organic composition is covalently ornon-covalently combined with the biologically active entities,substrate, and/or polymeric covering material. The biologicallycompatible organic composition interacts with the biologically activeentities and reactive chemical groups of the substrate material and/orcovering material to prevent the biologically active entities fromloosing biological activity under conditions that would otherwisesignificantly degrade the biological activity of the entities. Theseconditions include sterilization and storage. With expandableendoluminal medical devices, for example, mechanical compaction andexpansion of such devices can also significantly degrade the biologicalactivity of the entities.

In some cases, the additional biologically compatible organiccomposition seems to maintain the biological activity of the entities,particularly during immobilization, sterilization, storage, and/ormechanical manipulation by limiting undesirable alterations to theentities often induced by immobilization, sterilization, storage, and/ora mechanical manipulation process. The activity-diminishing alterationscould include conformational changes to a biologically active entityobscuring an active site on the entity. The activity-diminishingalterations could also include interactions between neighboringimmobilized biologically active entities. Rearrangements of immobilizedbiologically active entities with respect to a polymeric coveringmaterial are other possible activity-diminishing alterations to theentities. Simple denaturation, or other degradation, of the immobilizedbiologically active entities could be another means by which theentities loose biological activity. As described in greater detailherein, biologically active entities immobilized, sterilized, stored,and/or mechanically manipulated in the presence of the additionalbiologically compatible organic composition may retain a degree ofbiological activity significantly greater than a similar immobilizedbiologically active entity processed under the same conditions in theabsence of the additional biologically compatible organic composition.

The additional biologically compatible organic composition can beremoved from a sterilized medical device during post-sterilizationprocessing or the composition can be removed by physiological processesof an implant recipient following deployment of the sterilized medicaldevice at an implantation site.

Preferred biologically active entities reduce or inhibit thrombusformation on surfaces of a substrate and/or covering material.Glycosaminoglycans are preferred anti-thrombotic agents for use in thepresent invention, with dermatan disulfate, dermatan disulfate analogs,and derivatives being particularly preferred. Other preferredbiologically active substances reduce undesirable cellular growth fromtissue in which the present invention is implanted. Preferredanti-proliferative agents for use in the present invention include, butare not limited to, dexamethasone, rapamycin, and paclitaxel.

Accordingly, one embodiment of the present invention relates to amedical device comprising a substrate material, a polymeric coveringmaterial attached to at least a portion of a surface of said substratematerial, a plurality of biologically active entities having heparincofactor II binding activity covalently attached to at least a portionof said polymeric covering material, and wherein said biologicallyactive entities have a heparin cofactor II binding activity of at least5 picomoles heparin cofactor II per square centimeter (pmol/cm²). Inother embodiments, the anti-thrombin binding activity is at least 12picomoles heparin cofactor II per square centimeter (pmol/cm²) substratematerial, or at least 20 picomoles heparin cofactor II per squarecentimeter (pmol/cm²) substrate material. In some embodiments, theheparin cofactor II binding activity is at least 50 pmol/cm² picomolesheparin cofactor II per square centimeter (pmol/cm²) substrate material.

In another embodiment, the invention relates to a medical devicecomprising a substrate material, a polymeric covering material attachedto at least a portion of a surface of said substrate material, aplurality of biologically active entities having heparin cofactor IIbinding activity of at least 5 picomoles heparin cofactor II per squarecentimeter (pmol/cm²) covalently attached to at least a portion of saidpolymeric covering material, and a biologically compatible compositioncombined with said polymeric covering material wherein said biologicallycompatible composition has antithrombin III binding activity.

In another embodiment, the present invention relates to a medical devicecomprising a substrate material, a polymeric covering material attachedto at least a portion of a surface of said substrate material, a firstplurality of biologically active entities having heparin cofactor IIbinding activity and a second plurality of biologically active entitieshaving anti-thrombin III binding activity covalently attached to atleast a portion of said polymeric covering material, and a biologicallycompatible composition combined with said polymeric covering material,wherein said biologically active entities have a heparin cofactor IIbinding activity of at least 5 picomoles heparin cofactor II per squarecentimeter (pmol/cm²) and at least 5 picomoles anti-thrombin III persquare centimeter.

In yet another embodiment, the present invention relates to a medicaldevice comprising a substrate material, a plurality of chemical entitieshaving heparin cofactor II binding activity present on at least aportion of said substrate material, a first biologically compatiblecomposition combined with said substrate material, and a secondbiologically compatible composition admixed therewith.

A further embodiment of the present invention relates to a medicaldevice comprising a substrate material, a polymeric covering materialattached to at least a portion of a surface of said substrate material,a plurality of chemical entities having heparin cofactor II bindingactivity present on at least a portion of said polymeric coveringmaterial, a first biologically compatible composition combined with saidsubstrate material, and a second biologically compatible compositionadmixed therewith.

In embodiments relating to non-covalently combined biologicallycompatible organic compositions, at least a portion of the organiccomposition is often released from the sterilized or mechanicallymanipulated medical device within several hours when placed in a 0.15 Mphosphate buffer solution having a temperature of about thirty-sevendegrees centigrade and a substantially neutral pH. Presence of thereleased compounds can be detected in the buffer solution with routineassay techniques.

In embodiments relating to covalently combined biologically compatibleorganic compositions, the organic composition is substantially retainedon the sterilized or mechanically manipulated medical device followingsterilization or mechanical manipulation.

In yet other embodiments, the covalently combined biologicallycompatible organic composition may be released from the polymericcovering material through reversal of a covalent bond. Presence of thecompounds released by reversal of a covalent bond can be detected in abuffer solution with routine assay techniques.

In some embodiments, the biologically compatible organic composition maybe admixed prior to mechanical manipulation and/or sterilization. Inother embodiments, the biologically compatible organic composition maybe admixed following mechanical manipulation or sterilization (i.e., inan operating room). This is particularly useful when the organiccomposition may be degraded through mechanical manipulation orsterilization of a substrate or device utilizing the composition. Thisalso permits the organic composition to be placed at particularlocations on a substrate or device and at varying dosages.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a polymeric substrate materialhaving a multiplicity of reactive chemical groups thereon.

FIG. 1A is a schematic representation of a metallic substrate material.

FIG. 2 is a schematic representation of a polymeric substrate materialhaving a plurality of biologically active entities immobilized thereto.

FIG. 3 is a schematic representation of a polymeric substrate materialhaving a polymeric covering material with a multiplicity of reactivechemical groups thereon.

FIG. 3A is a schematic representation of a metallic substrate materialhaving a polymeric covering material with a multiplicity of reactivechemical groups thereon.

FIG. 4 is a schematic representation of a polymeric substrate materialhaving a polymeric covering material with a plurality of biologicallyactive entities immobilized thereto.

FIG. 4A is a schematic representation of a metallic substrate materialhaving a polymeric covering material with a plurality of biologicallyactive entities immobilized thereto.

FIG. 5 is a schematic representation of a polymeric substrate materialhaving a plurality of biologically active entities immobilized theretoand an additional biologically compatible composition combinedtherewith.

FIG. 6 is a schematic representation of a polymeric substrate materialhaving a polymeric covering material with a plurality of biologicallyactive entities immobilized thereto and an additional biologicallycompatible composition combined therewith.

FIG. 6A is a schematic representation of a metallic substrate materialhaving a polymeric covering material with a plurality of biologicallyactive entities immobilized thereto and an additional biologicallycompatible composition combined therewith.

FIG. 6B is a schematic representation of a polymeric substrate materialhaving a polymeric covering material with a plurality of biologicallyactive entities immobilized thereto showing some of the biologicallycompatible composition illustrated in FIG. 6 having been released fromthe substrate material and polymeric covering material.

FIG. 6C is a schematic representation of a metallic substrate materialhaving a polymeric covering material with a plurality of biologicallyactive entities immobilized thereto showing some of the biologicallycompatible composition illustrated in FIG. 6A having been released fromthe substrate material and polymeric covering material.

FIG. 7 is a schematic representation of a polymeric substrate materialhaving three layers of polymeric covering material applied thereto witha plurality of biologically active entities immobilized thereto and anadditional biologically compatible composition combined therewith.

FIG. 7A is a schematic representation of a metallic substrate materialhaving three layers of polymeric covering material applied thereto witha plurality of biologically active entities immobilized thereto and anadditional biologically compatible composition combined therewith.

FIG. 7B is a schematic representation of a polymeric substrate materialhaving three layers of polymeric covering material applied thereto witha plurality of biologically active entities immobilized thereto showingsome of the biologically compatible composition illustrated in FIG. 7having been released from the substrate material and polymeric coveringmaterial.

FIG. 7C is a schematic representation of a metallic substrate materialhaving three layers of polymeric covering material applied thereto witha plurality of biologically active entities immobilized thereto showingsome of the biologically compatible composition illustrated in FIG. 7Ahaving been released from the substrate material and polymeric coveringmaterial.

FIG. 8 is a bar graph illustrating how sterilization of unbound heparindoes not significantly reduce the biological activity of the heparin.

FIG. 9 is a bar graph illustrating the effect of a variety ofbiologically compatible organic compositions on the biological activityof end-point attached heparin immobilized to reactive chemical groups ona polymeric covering material during and after exposure of theimmobilized heparin to an ethylene oxide sterilization regimen.

FIG. 10 is a bar graph illustrating the ability of added heparin ordextran sulfate biologically compatible organic compositions to resultin high levels of ATIII binding activity of heparin immobilized to apolymeric covering material on a substrate during and after exposure ofthe immobilized heparin to an ethylene oxide sterilization regimen.

FIG. 11 is a bar graph illustrating the ability of added dextran sulfateto maintain the biological activity of end-point attached heparinimmobilized on a polyvinyl alcohol coated substrate during and afterexposure of the immobilized heparin to an ethylene oxide sterilizationregimen.

FIG. 12 is a bar graph illustrating the ability of added glycerol tomaintain the biological activity of end-point attached heparinimmobilized on a polymeric covering material of a substrate followingcompaction and expansion of the substrate material.

FIG. 13 is a bar graph illustrating the ability of added glycerol andheparin to maintain the biological activity of end-point attachedheparin immobilized on a polymeric covering material of a substratefollowing mechanical compaction, exposure to an ethylene oxidesterilization regimen, and mechanical expansion of the substratematerial.

FIG. 14 is a schematic representation of a polymeric substrate materialhaving a polymeric covering material with a plurality of biologicallyactive entities immobilized thereto and reactive chemical groupsthereon.

FIG. 15 is a schematic representation of a metallic substrate materialhaving a polymeric covering material with a plurality of biologicallyactive entities immobilized thereto and reactive chemical groupsthereon.

FIG. 16 is a schematic representation of a polymeric substrate materialhaving a polymeric covering material with a plurality of biologicallyactive entities and an additional biologically compatible compositioncovalently combined thereto.

FIG. 17 is a schematic representation of a metallic substrate materialhaving a polymeric covering material with a plurality of biologicallyactive entities and an additional biologically compatible compositioncovalently combined thereto.

FIG. 18 is a schematic representation of embodiments of the presentinvention having a second biologically compatible composition combinedtherewith.

FIG. 19 is a schematic representation of embodiments of the presentinvention having a second biologically compatible composition combinedtherewith.

FIG. 20 is a schematic representation of a polymeric substrate materialhaving a polymeric covering material with a plurality of biologicallyactive entities immobilized thereto, a first biologically compatiblecomposition, and a second biologically compatible composition combinedtherewith.

FIG. 21 is a schematic representation of a metallic substrate materialhaving a polymeric covering material with a plurality of biologicallyactive entities immobilized thereto, a first biologically compatiblecomposition, and a second biologically compatible composition combinedtherewith.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to materials and devices with biologicallyactive entities having heparin cofactor II binding activity immobilizedthereto. The biologically active entities retain significant biologicalactivity following immobilization, sterilization, mechanical compactionand expansion, and/or storage conditions that would otherwisesignificantly decrease the heparin cofactor II binding activity of theimmobilized entities. The biological activity of an immobilizedbiological entity subjected to such conditions may be positivelyinfluenced by the presence of at least one additional biologicallycompatible composition covalently or non-covalently combined with thebiologically active entities. In most embodiments, the additionalcomposition is an organic compound. In some embodiments, however, thebiologically compatible composition is an inorganic compound. Inpreferred embodiments, the additional composition is a carbohydrate inthe form of a polysaccharide. Preferred polysaccharides areglycosaminoglycans. Preferred glycosaminoglycans are heparincompositions, heparin analogs, heparin derivatives, dermatan disulfate,dermatan disulfate analogs, and dermatan disulfate derivatives.

Referring to FIGS. 1 and 2, some polymeric substrate materials (12) havemultiplicities of reactive chemical groups (16) populating at least aportion of the surfaces of the substrate materials to which a pluralityof biologically active entities (17) are attached, confined, orotherwise immobilized. Most biologically active entities (17) arecovalently attached, or bound, to the substrate materials (12) throughthe reactive chemical groups (16). Surfaces of the polymeric substratematerial (12) can be smooth, rough, porous, curved, planar, angular,irregular, or combinations thereof. In some embodiments, substratematerials with surface pores have internal void spaces extending fromthe porous surface of the material into the body of the material. Theseporous substrate materials have internal substrate material bounding thepores that often provides surfaces amenable to immobilizing biologicallyactive entities. Whether porous or non-porous, substrate materials canbe in the form of filaments, films, sheets, tubes, meshworks, wovens,non-wovens, and combinations thereof.

Suitable substrate materials (12) for immobilizing biologically activeentities (17) include biocompatible polymeric materials such aspolyethylene, polyurethane, silicone, polyamide-containing polymers, andpolypropylene. Full density or porous polytetrafluoroethylene is asuitable polymeric substrate material (12) if reactive chemical groups(16) are introduced in constituents of the polymeric material. Substratematerials with a multiplicity of reactive chemical groups that are partof the substrate material are referred to herein as “functionalizablematerials.” Following reaction of a biologically active entity with afunctionalizable substrate material, the substrate material isconsidered functionalized and the biologically active entityimmobilized. In order to maintain the biological activity of theimmobilized entity during subsequent processing conditions, such assterilization, mechanical compaction and expansion, or storage, anadditional biologically compatible organic chemical composition isnon-covalently combined with the functionalized material and immobilizedentity.

Substrate materials can also have a multiplicity of reactive chemicalgroups added to surfaces of the materials through the application of oneor more covering compositions, or materials, to the surfaces. At least aportion of a covering material has chemical elements, groups, compounds,or components that are reactive to biologically active entities andserve to attach, confine, or otherwise immobilize a biologically activeentity in a biologically active form to the covering material. Thecovering material can be applied in the form of a solute, particle,dispersion, coating, or overlay and attached to the substrate materialin a variety of ways including, but not limited to, covalent bonding,adsorption, such as, physisorption or chemisorption, and non-covalentbonding, such as hydrogen bonding or ionic bonding. In preferredembodiments, the covering material is applied in a solution and forms acontinuous or discontinuous film layer on one or more surfaces of thesubstrate material upon removal of the solvent. The covering materialcan be applied in one or more layers. The chemical constituents of thecovering material in each layer can be the same or different. In someembodiments, the covering material is cross-linked to itself or othercovering materials in other layers. The cross-linking bonds can becovalent or ionic.

Substrate materials (12, 14) lacking reactive chemical groups on theirsurfaces (FIG. 1A) (or lacking appropriately reactive chemical groups)are covered, at least in part, with a polymeric covering material (18)having a multiplicity of reactive chemical groups (16) thereon (FIGS. 3and 3A) to which biologically active entities (17) can be attached,confined, or otherwise immobilized (FIGS. 4 and 4A). Most biologicallyactive entities (17) are covalently attached, or bound, to the polymericcovering material (18) through the reactive chemical groups (16) of thecovering material (18). The polymeric covering material (18) forms atleast one layer on at least a portion of a substrate material (12, 14).In some embodiments, the polymeric covering material (18) iscross-linked (19) to itself or other layers (18 a, 18 b) of polymericcovering material (FIGS. 7 and 7 a). The cross-linking can be covalent,ionic, or both. Substrate materials amenable to covering are glass,metals (14), ceramics, polymeric materials (12), particularly chemicallyinert polymeric materials such as polytetrafluoroethylene.

At least one type of biologically active entity having heparin cofactorII or anti-thrombin III binding capability (17) is chemically attached,confined, or otherwise immobilized to suitable reactive chemical groups(16) on the substrate material (12, 14) and/or covering material (18).

Biologically compatible compositions (11, 15, 100) include, but are notlimited to, antithrombotics, anticoagulants, fibrinolytic orthrombolytic agents, antibiotics, antimicrobial/antiseptic compounds,anti-viral compounds, anti proliferatives, cell adhesive compounds, cellanti-adhesive compounds, and anti-inflammatories. Antithrombotics ofparticular interest are glycosaminoglycans, particularly dermatandisulfate, dermatan disulfate derivatives and analogs, heparin, andheparin derivatives and analogs. Other anticoagulant agents include, butare not limited to, hirudin, activated protein C and prostaglandins.Fibrinolytic or thrombolytic agents include, but are not limited to,streptokinase, urokinase, and tissue plasminogen activator (tPA).Examples of antibiotics include, but are not limited to, penicillin,tetracycline, chloramphenicol, minocycline, doxycycline, vancomycin,bacitracin, kanamycin, neomycin, gentamycin, erythromycin andcephalosporins. Examples of cephalosporins include cephalothin,cephapirin, cefazolin, cephalexin, cephradine, cefadroxil, cefamandole,cefoxitin, cefaclor, cefuroxime, cefonicid, ceforanide, cefotaxime,moxalactam, ceftrizoxime, ceftriaxone, and cefoperazone. Examples ofantimicrobial/antiseptics include, but are not limited to, silversulfadiazine, chlorhexidine, peracetic acid, sodium hypochlorite,triclosan, phenols, phenolic compounds, iodophor compounds, quaternaryammonium compounds, chlorine compounds, heparin and combinationsthereof. Examples of anti-viral agents include, but are not limited to,alpha.-methyl-1-adamantanemethylamine, hydroxy-ethoxymethylguanine,adamantanamine, 5-iodo-2′-deoxyuridine, trifluorothymidine, interferon,and adenine arabinoside. Cell adhesive compounds include, but are notlimited to, fibronectin, laminin, collagen, vitronectin, osteopontin,RGD peptides, RGDS peptides, YIGSR peptides, and antibodies targetingcell surface antigens. Compounds that may resist cellular attachmentinclude Poly HEMA, poly ethylene glycol, polysaccharides,polyvinylpyrrolidone, and phospholipids. Other biologically activeentities include, but are not limited to, enzymes, organic catalysts,ribozymes, organometallics, proteins, glycoproteins, peptides, polyaminoacids, antibodies, nucleosides, nucleotides, nucleic acids, steroidalmolecules, antibiotics, antimicrobial compounds, antimycotics,cytokines, carbohydrates, oleophobics, lipids, pharmaceuticals, andtherapeutics.

While a variety of biologically active entities (17) can be used in thepresent invention, as described above, entities capable of interactingwith components of mammalian blood to prevent coagulation or thrombusformation on surfaces of a substrate material (12, 14) or coveringmaterial (18) by the blood components are most preferred. Many of thesebiologically active entities are oligosaccharides or polysaccharides.Some of the polysaccharides are glycosaminoglycans including glucosamineor galactosamine compositions. Preferred glycosaminoglycans are heparincompositions, heparin analogs, heparin derivatives, dermatan disulfate,dermatan disulfate analogs, and dermatan disulfate derivatives. Heparinis a complex glycosaminoglycan with many biological functions mediatedby its binding to growth factors, enzymes, morphogens, cell adhesionsmolecules, and cytokines. The biological activity of heparin to functionas an anticoagulant is based on the ability of heparin to act as acatalyst for thrombin and antithrombin III binding. Most of theanti-coagulant activity of heparin is associated with a pentasaccharidesequence that facilitates this binding. Another complexglycosaminoglycan is dermatan disulfate that also has many biologicalfunctions, and biological activity of an anticoagulant based on itsability to act as a catalyst for the inhibition of thrombin by heparinco-factor II (HC II). Dermatan disulfate may be produced from dermatansulfate in a synthetic reaction disclosed in U.S. Pat. No. 5,922,690,which is incorporated herein by reference. Additional disclosure ofdermatan disulfate synthesis is found in U.S. Pat. No. 5,705,493, whichis incorporated herein by reference. Heparin may be modified by chemicalprocesses, substantially similar to those described for dermatandisulfate, to obtain an over sulfated heparin derivative with HCIIactivity.

The most preferred polysaccharide composition for immobilization in thepresent invention is a polysaccharide composition having a free terminalaldehyde group made according the teachings of U.S. Pat. No. 4,613,665,issued to Larm, which is incorporated herein by reference. The mostpreferred polysaccharide for use is a dermatan disulfate made accordingto U.S. Pat. No. 5,922,690, which is incorporated herein by reference.In the process of making dermatan disulfate with a free terminalaldehyde group, the dermatan disulfate is subjected to degradation bydiazotation to form a fragment having a free terminal aldehyde group.The free terminal aldehyde group allows the dermatan disulfatecomposition to be “end point attached” to primary amino groups of asubstrate or polymeric covering material to form an imine which, byreduction, is converted to a secondary amine. End point attachment ofthe dermatan disulfate composition permits the dermatan disulfate to beimmobilized in a conformation that most advantageously exposes thebiologically active portion of the dermatan disulfate composition tocomponents of the blood responsible for coagulation and thrombusformation. When exposed to the blood components responsible for thrombusformation and coagulation, the optimally immobilized dermatan disulfateinteracts with the blood components to reduce or prevent thrombusformation or other coagulation events on surfaces of the substrateand/or covering material.

Other desirable biologically active entities (17) for use in the presentinvention include heparin and synthetic heparin compositions referred toas “fondaparinux,” compositions involving antithrombin III-mediatedinhibition of factor Xa, entities catalyzing HC II binding withthrombin, antiproliferatives, and anti-inflammatories.

Despite an optimized immobilization scheme, the biological activity of adermatan disulfate-based biological entity is significantly decreasedduring sterilization, mechanical compaction and expansion, and/orstorage of the entities (FIGS. 9, 11, 12, and 13). As discussed above,the decrease in biological activity of an immobilized biologicallyactive entity may be caused by a variety of factors. Regardless of themechanism by which the biological activity of an immobilized entity isdecreased, addition of a biologically compatible organic compositioncovalently and/or non-covalently combined with the immobilizedbiologically active entity may maintain the biological activity of theentity during and after sterilization, mechanical manipulation—such asmechanical compaction and expansion, and/or storage of the entities.

The additional biologically compatible organic composition can havebiological activity or no biological activity. The additionalbiologically compatible organic composition can be a carbohydrate in theform of polyhydroxy aldehydes or ketones and their derivatives. Thesecarbohydrates include monosaccharides, disaccharides, oligosaccharides,and polysaccharides, including glycosaminoglycans, glycosaminomannans,and storage polysaccharides such as dextran and its derivatives. Otherbiologically compatible organic compositions suitable for use in thepresent invention include acid mucopolysaccharides, amino acids,polypeptides, proteins, glycoproteins, nucleosides, nucleotides,polynucleotides, or other biologically compatible aliphatic or aromaticcompound, charged or uncharged, having a molecular weight less thanabout 100,000 MW.

Referring to FIGS. 5-6A, covered or uncovered substrate materials (14,12, respectively) having biologically active entities (17) immobilizedthereon have an additional biologically compatible composition (100)combined with the biologically active entities (17), the substratematerial (12, 14) and/or the covering material (18). The biologicallycompatible composition is preferably organic. The biologicallycompatible organic composition can be applied to the immobilizedbiologically active entities, substrate, and/or covering material in avariety of ways. In a preferred embodiment, a suitablecarbohydrate-based biologically compatible composition is dissolved inan aqueous solvent and the solution applied to the immobilizedbiologically active entities, substrate, and/or polymeric coveringmaterial by spraying, dip coating, immersing, rolling, spreading, orother deposition means. In appropriate systems, biologically compatiblecompositions can be dissolved in organic solvents and similarly applied.

The preferred embodiment of the present invention relates to asterilized medical device for implantation, or other placement, at ananatomical site. Most preferred are sterilized medical devices forplacement inside an anatomical structure delimiting a void space, orlumen, to reinforce the anatomical structure or maintain the void spacedelimited thereby. When these sterilized devices are used within avascular structure, immobilized biologically active entities in the formof end point attached heparin interact with blood flowing through, oraround, the devices to minimize or prevent formation of thrombus orother coagulation products on blood-contacting surfaces of the devices.In a preferred embodiment, the additional biologically compatibleorganic composition is a polyethylene glycol compound covalentlycombined with the substrate material and/or covering material. Thecovalently bound heparin is allowed to remain with the sterilizeddevices. The preferred sterilization method includes ethylene oxide gas.

The manufacturing of medical devices may require mechanical manipulationthat often reduces the biological activity of an immobilizedbiologically active entity. The additional biologically compatiblecomposition combined with the immobilized biologically active entities,substrate material, and/or covering material as described above, mayalso maintain the biological activity of the immobilized biologicallyactive entities following mechanical compaction and expansion of amedical device (FIGS. 12 and 13). Expandable stents and stent-grafts aremedical devices for which improved biological activity of immobilizedbiologically active entities is particularly significant.

The present invention, therefore, provides sterilized devices havingbiologically active entities immobilized thereto where the biologicalactivity of the immobilized entities is significantly retained duringand after a sterilization process (FIGS. 9-11, and 13). Prior tosterilization, the devices can be mechanically manipulated, throughcompaction and expansion, for example, and retain significant biologicalactivity (FIGS. 12 and 13).

FIG. 14 schematically illustrates embodiments of the present invention(50) having a polymeric substrate (12) having a polymeric covering, orcoating, material (18) cross-linked (19) thereon. Covering (18) has aplurality of immobilized biologically active entities “B” (17) attachedthereto. Covering (18) also has a plurality of chemically reactivegroups “R” (13) thereon to which a biologically compatible composition“S” (15) can be covalently attached (FIGS. 16 and 17). In someembodiments, the covalent bonds are reversible thereby rendering thebiologically compatible composition “S” (15) releasable from theinvention under appropriate conditions. FIGS. 15 and 17 schematicallyillustrate similar constructions (50) using a metallic substrate (14).

FIGS. 18 and 19 schematically illustrate embodiments of the presentinvention (70) having a polymeric substrate (12) or metallic substrate(14) having a polymeric covering, or coating, material (18) cross-linked(19) thereon. Covering (18) has a plurality of immobilized biologicallyactive entities “B” (17) and a first biologically compatible composition“S” (15) covalently attached thereto. In some embodiments, the covalentbonds are reversible thereby rendering the biologically compatiblecomposition “S” (15) releasable from the invention under appropriateconditions. In addition, this embodiment has a second biologicallycompatible composition “A” (11) admixed therewith.

FIGS. 20 and 21 schematically illustrate embodiments of the presentinvention (80) having a polymeric substrate (12) or metallic substrate(14) having a polymeric covering, or coating, material (18) cross-linked(19) thereon. Covering (18) has a plurality of biologically activeentities “B” (17) immobilized thereto. A first biologically compatiblecomposition (100) is combined with the biologically active entities(17). In addition, this embodiment has second biologically compatiblecomposition “A” (11) admixed therewith.

EXAMPLES

Calculations of heparin activity on surfaces in the present inventionwere conducted using the surface area of only one side of the samplematerial, although the entire sample, including interstices, may haveheparin immobilized thereon. The heparin activity was assayed bymeasuring the ability, or capacity, of the end-point attached heparin tobind a known quantity of anti-thrombin III (ATIII). The results wereexpressed as picomoles anti-thrombin III (ATIII) bound per squarecentimeter of substrate material (pmol ATIII/cm² substrate material).This assay is described by Larsen M. L., et al. in “Assay of plasmaheparin using thrombin and the chromogenic substrateH-D-Phe-Pip-Arg-pNA” (S-2238) (Thromb. Res. 1978; 13:285-288) andPasche, et al. in “A binding of antithrombin to immobilized heparinunder varying flow conditions” (Artif. Organs 1991; 15:281-491).

ATIII binding activity per surface area of substrate material is definedas the number of picomoles of ATIII bound per apparent surface area ofcovered or uncovered substrate material. The apparent substrate surfacearea does not take into account multiple covered surfaces nor porosityconsiderations of a porous substrate material. If the substrate materialis porous, the effect of porosity on surface area is not considered forthese calculations. For example, the apparent surface area of acylindrical tubular ePTFE vascular graft (which is made of a porousmaterial) with end-point attached heparin immobilized on substratematerial comprising the inner surface of the tubular graft is calculatedas it is for any cylindrical geometry as 2πrL: where r is the graftinner radius; L is the axial length; and π is the number pi. It isimportant to note that the porous nature of ePTFE and its effect onsurface area is not accounted for herein. Accordingly, non-poroussubstrate materials that are cut into squares for analysis are taken tohave a surface area of the length multiplied by the width.

Calculations of dermatan disulfate activity on surfaces in the presentinvention were conducted using the surface area of only one side of thesample material, although the entire sample, including interstices, mayhave dermatan disulfate immobilized thereon. The dermatan disulfateactivity was assayed by measuring the ability, or capacity, of theend-point attached dermatan disulfate to bind a known quantity ofheparin cofactor II (HC II). The results were expressed as picomolesheparin cofactor II (HC II) bound per square centimeter of substratematerial (pmol HC II/cm² substrate material). Samples approximately onesquare centimeter (1 cm²) in size are cut from the construction andassayed for dermatan disulfate activity by measuring the capacity of theend point attached dermatan sulfate to bind heparin cofactor II (HCII).The measurement of dermatan disulfate activity is similar to thatdescribed previously for heparin activity by Larsen M. L., et al., in“Assay of plasma heparin using thrombin and the chromogenic substrateH-D-Phe-Pip-Arg-pNA (S-2238).” Thromb Res 13:285-288 (1978) and PascheB., et al., in “A binding of antithrombin to immobilized heparin undervarying flow conditions.” Artif. Organs 15:281-491 (1991). For thedermatan disulfate activity assay, HCII is allowed to bind to thedermatan disulfate surface, eluted from the surface by an excess ofsoluble dermatan disulfate, and combined with thrombin in a colorimetricassay for thrombin activity. The assay indirectly determines the amountof HCII present by measuring HCII-mediated inhibition of human thrombin.The amount of HCII is determined from a standard curve derived by mixingknown amounts of dermatan disulfate, HCII, thrombin, and a syntheticthrombin substrate (known as an amidolytic assay). A similar approachfor measuring soluble dermatan sulfate activity has been previouslydescribed by Dupouy D., et al., in “A simple method to measure dermatansulfate at sub-microgram concentrations in plasma.” Thromb. Haemost.60:236-239 (1988). The results are expressed as amount of HCII bound perunit surface area substrate material in picomoles per square centimeter(pmol/cm2). All samples are maintained in a wet condition throughout theassay. It is important to note that while the approximately one squarecentimeter (1 cm²) samples each have a total surface area of two squarecentimeters (2 cm²) if both sides of the material are considered, onlyone surface on the sample (i.e., 1 cm²) is used for calculatingHCII-dermatan disulfate binding activity in pmol/cm².

In an alternative method, dermatan disulfate activity is directlyquantified by measuring the amount of radiolabeled HCII bound to thedermatan disulfate-immobilized construct. This technique is similar tomethods described for measuring antithrombin III binding to immobilizedheparin constructs by Du Y. J., et al., in “Protein adsorption onpolyurethane catheters modified with a novel antithrombin-heparincovalent complex.” J. Biomed. Mater. Res. 80A:216-225 (2007). Thedermatan disulfate construct is incubated with a solution of HCII thathas been covalently labeled with the radioisotope Iodine-125 (¹²⁵I).After incubation the surface is repeatedly rinsed and the amount ofradiation emitted from the construct is measured by a gamma counter.Because the ratio of emission to HCII mass is known, the amount of HCIIcan be determined. The results are expressed as amount of HCII bound perunit surface area substrate material in picomoles per square centimeter(pmol/cm²).

HC II binding activity per surface area of substrate material is definedas the number of picomoles of HC II bound per apparent surface area ofcovered or uncovered substrate material. The apparent substrate surfacearea does not take into account multiple covered surfaces nor porosityconsiderations of a porous substrate material. If the substrate materialis porous, the effect of porosity on surface area is not considered forthese calculations. For example, the apparent surface area of acylindrical tubular ePTFE vascular graft (which is made of a porousmaterial) with end-point attached dermatan disulfate immobilized onsubstrate material comprising the inner surface of the tubular graft iscalculated as it is for any cylindrical geometry as 2πrL: where r is thegraft inner radius; L is the axial length; and π is the number pi. It isimportant to note that the porous nature of ePTFE and its effect onsurface area is not accounted for herein. Accordingly, non-poroussubstrate materials that are cut into squares for analysis are taken tohave a surface area of the length multiplied by the width.

Example 1

This example demonstrates retention of biological activity of unbound“neat” heparin following exposure of the heparin to an ethylene oxide(EtO) sterilization process.

In this example, unsterilized USP grade heparin-sodium in lyophilizedpowder form was obtained from Celsus Laboratories (Cincinnati, Ohio).Measured quantities of heparin were placed into CHEX-ALL® sterilizationpouches (Long Island City, N.Y.) for testing. One group ofheparin-containing pouches was exposed to EtO sterilization. Ethyleneoxide sterilization was carried out under conditions of conditioning forone hour (1 hr), an EtO gas dwell time of one hour (1 hr), a set pointtemperature of fifty-five degrees centigrade (55° C.), and an aerationtime of twelve hours (12 hr). Another group was subjected to thesterilization procedure in the absence of EtO. A third group was notexposed to the sterilization procedure.

Following the sterilization procedure, known quantities of heparin wereremoved from each pouch and tested for bio-activity with an ACTICHROMEHeparin (anti-FXa) assay kit available from American Diagnostica Inc.(Stamford, Conn.). Bioactivity values for each heparin sample wereexpressed as international units of heparin per mass of heparin (IU/mg).International units of heparin are calculated based on Factor X_(a)inactivation by ATIII that is catalyzed by heparin. International unitsare therefore a measure of the ATIII binding activity of heparin. Anyreduction in heparin activity is expressed simply as a reduction in theIU/mg for comparable heparin controls from the ACTICHROME test. Heparinexhibiting a reduction in activity is considered to have beendeactivated to a degree by the sterilization process.

FIG. 8 is a bar graph illustrating the effect of EtO sterilization onthe anti-thrombin III (ATIII) binding activity of dry powdered heparinin an unbound state. FIG. 8 shows the mean activity levels, expressed asIU/mg, for the heparin samples (n=3) in each group. Control heparinsamples that did not undergo sterilization had a mean value of 138IU/mg. Control heparin samples that underwent the sterilization processin the absence of EtO (i.e., high humidity, high temperatures, etc.) hada mean value of 119 IU/mg. The heparin samples that underwent thesterilization process in the presence of EtO had a mean value of 123IU/mg. The heparin samples exposed to the sterilization process in theabsence of EtO had an fourteen percent (14%) decrease in activitycompared to the unsterilized control samples, while the samples exposedto the sterilization process in the presence of EtO had only an elevenpercent (11%) decrease in activity. As seen from FIG. 8, sterilizationof unbound, neat, heparin powder in the presence or absence of EtO doesnot significantly reduce ATIII binding to the heparin when compared tounsterilized control samples. The anti-thrombin III binding activity ofunbound, unsterilized, heparin is not significantly diminished bysterilization without EtO or sterilization with EtO. Therefore,degradation of the anti-thrombin III binding activity of immobilizedheparin subjected to similar EtO sterilization conditions must be causedby a mechanism other than simple exposure to sterilization with orwithout EtO.

Example 2

This example describes the construction of an embodiment of the presentinvention in which heparin anti-thrombin III (ATIII) binding is notsignificantly diminished by exposure to EtO sterilization.

In accordance with U.S. Pat. No. 6,653,457, which is incorporated hereinby reference, an aldehyde modified heparin composition made according toU.S. Pat. No. 4,613,665, which is incorporated herein by reference, wasend-point attached to a covering material, or coating layer, placed onan expanded polytetrafluoroethylene (ePTFE) material. An additionalbiologically compatible organic composition was incorporated within thecovering material and bound heparin to enable the immobilized heparin toundergo EtO sterilization without significant loss in biologicalactivity.

An ePTFE material in sheet form was obtained from W.L. Gore &Associates, Inc., Flagstaff, Ariz. under the tradename GORE™Microfiltration Media (GMM-406). A covering material in the form of abase coating was applied to the ePTFE material by mounting the materialon a ten centimeter (10 cm) diameter plastic embroidery hoop andimmersing the supported ePTFE material first in 100% isopropyl alcohol(IPA) for about five minutes (5 min) and then in a solution of LUPASOL®polyethylene imine (PEI) and IPA in a one to one ratio (1:1). LUPASOL®water-free PEI was obtained from BASF and diluted to a concentration ofabout four percent (4%) and adjusted to pH 9.6. Following immersion ofthe ePTFE material in the solution for about fifteen minutes (15 min),the material was removed from the solution and rinsed in deionized (DI)water at pH 9.6 for fifteen minutes (15 min). PEI remaining on the ePTFEmaterial was cross-linked with a 0.05% aqueous solution ofglutaraldehyde (obtained from Amresco) at pH 9.6 for fifteen minutes (15min). Additional PEI was added to the construction by placing theconstruction in a 0.5% aqueous solution of PEI at pH 9.6 for fifteenminutes (15 min) and rinsing again in DI water at pH 9.6 for fifteenminutes (15 min). The imine formed as a result of the reaction betweenglutaraldehyde and the PEI layer is reduced with a sodiumcyanborohydride (NaCNBH₃) solution (5 g dissolved in 1 L DI water, pH9.6) for fifteen minutes (15 min) and rinsed in DI water for thirtyminutes (30 min).

An additional layer of PEI was added to the construction by immersingthe construction in 0.05% aqueous glutaraldehyde solution at pH 9.6 forfifteen minutes (15 min), followed by immersion in a 0.5% aqueoussolution of PEI at pH 9.6 for fifteen minutes (15 min). The constructionwas then rinsed in DI water at pH 9.6 for fifteen minutes (15 min). Theresultant imines were reduced by immersing the construction in asolution of NaCNBH₃ (5 g dissolved in 1 L DI water, pH 9.6) for fifteenminutes (15 min) followed by a rinse in DI water for thirty minutes (30min). A third layer was applied to the construction by repeating thesesteps. The result was a porous hydrophobic fluoropolymeric base materialhaving a hydrophilic cross-linked polymer base coat on substantially allof the exposed and interstitial surfaces of the base material.

An intermediate chemical layer was attached to the polymer base coat inpreparation for placement of another layer of PEI on the construction.The intermediate ionic charge layer was made by incubating theconstruction in a solution of dextran sulfate (Amersham PharmaciaBiotech) and sodium chloride (0.15 g dextran sulfate and 100 g NaCldissolved in 1 L DI water, pH 3) at 60° C. for ninety minutes (90 min)followed by rinsing in DI water for fifteen minutes (15 min).

A layer of PEI, referred to herein as a “capping layer” was attached tothe intermediate layer by placing the construction in a 0.3% aqueoussolution of PEI (pH 9) for about forty-five minutes (45 min) followed bya rinse in a sodium chloride solution (50 g NaCl dissolved in 1 L DIwater) for twenty minutes (20 min). A final DI water rinse was conductedfor twenty minutes (20 min).

Aldehyde modified heparin was end point attached, or conjugated, to thePEI layer(s) by placing the construction in a heparin-containing sodiumchloride salt solution (1.5 g heparin, 29.3 g NaCl dissolved in 1 L DIwater, pH 3.9) for one hundred twenty minutes (120 min) at sixty degreescentigrade (60° C.). A 2.86 mL volume of a 2.5% (w/v) aqueous NaCNBH₃solution was added to the one liter (1 L) heparin solution prior toadding the samples. The samples were then rinsed in DI water for fifteenminutes (15 min), borate buffer solution (10.6 g boric acid, 2.7 g NaOHand 0.7 g NaCl dissolved in 1 L DI water, pH 9.0) for twenty minutes (20min), and finally in DI water for fifteen minutes (15 min) followed bylyophilization of the entire construction to produce dry heparin boundto the ePTFE material. The presence and uniformity of the heparin wasdetermined by staining samples of the construction on both sides withtoluidine blue. The staining produced an evenly purpled surfaceindicating heparin was present and uniformly bound to the ePTFEmaterial.

By adding particular compounds or compositions to the heparin-boundconstruction, the biological activity of the heparin can be maintainedfollowing exposure to conditions that would otherwise decrease thebiological activity of the heparin. The conditions include, but are notlimited to, EtO sterilization, mechanical compaction and expansion, andstorage.

The above-described constructions coated with a covering material wereexposed to solutions of the following compounds to evaluate theirstabilizing effect on the biological activity of the heparin bound toparts of the coating: USP grade calcium chloride (Fisher Scientific),USP grade heparin sodium (Celsus), polyethylene glycol (20,000 molecularweight, Sigma), DEAE dextran (500,000 molecular weight, PK chemical),dextran sulfate sodium salt (8,000 molecular weight, Sigma), and dextran(9,500 molecular weight, Sigma) at concentrations of 0.5 g per 100 ml DIwater adjusted to pH 9.6. Dexamethasone was also utilized at 0.5 g per100 ml ethanol with no pH adjustment. Each of these solutions isreferred to herein as a “treatment solution.” The effect of thesevarious compounds on binding activity of heparin to anti-thrombin III(ATIII) following EtO sterilization was expressed as picomolesanti-thrombin III bound per square centimeter (cm²) substrate material.These data are summarized in FIG. 9.

To expose a particular heparin-containing construction to a particulartreatment solution, the construction was placed into a two liter (2 L)beaker and one hundred milliliters (100 ml) of treatment solution wasadded, sufficient to completely immerse the construction in thetreatment solution. Each construction was exposed to the treatmentsolution for one hour (1 hr) at sixty degrees centigrade (60° C.). Theconstruction was removed from the solution and lyophilized prior toexposure to a sterilization procedure.

In preparation for EtO sterilization, each lyophilized construction wasplaced and sealed in a Tower DUALPEEL® Self-Seal Pouch (AllegianceHealthcare Corp., McGaw Park, Ill.). Ethylene oxide sterilization wascarried out under conditions of conditioning for one hour (1 hr), an EtOgas dwell time of one hour (1 hr), a set point temperature of fifty-fivedegree centigrade (55° C.), and an aeration time of twelve hours (12hr).

After EtO sterilization, each construction (including controls) wasremoved from its pouch and washed in DI water for fifteen minutes (15min), borate buffer solution (10.6 g boric acid, 2.7 g NaOH and 0.7 gNaCl dissolved in 1 L DI water, pH 9.0) for twenty minutes (20 min), andfinally a rinse in DI water for fifteen minutes (15 min).

Samples approximately one square centimeter (1 cm²) in size were cutfrom the construction and assayed for heparin activity by measuring thecapacity of the end point attached heparin to bind ATIII. The assay isdescribed by Larsen M. L., et al., in “Assay of plasma heparin usingthrombin and the chromogenic substrate H-D-Phe-Pip-Arg-pNA (S-2238).”Thromb Res 13:285-288 (1978) and Pasche B., et al., in “A binding ofantithrombin to immobilized heparin under varying flow conditions.”Artif. Organs 15:281-491 (1991). The results were expressed as amount ofATIII bound per unit surface area substrate material in picomoles persquare centimeter (pmol/cm²). All samples were maintained in a wetcondition throughout the assay. It is important to note that while theapproximately one square centimeter (1 cm²) samples each have a totalsurface area of two square centimeters (2 cm²) if both sides of thematerial are considered, only one surface on the sample (i.e., 1 cm²)was used for calculating ATIII heparin-binding activity in pmol/cm².

FIG. 9 is a bar graph illustrating the effects various biologicallycompatible organic compositions non-covalently combined with heparinimmobilized on a covered substrate material on the anti-thrombin IIIbinding activity of the immobilized heparin following exposure of theimmobilized heparin to EtO sterilization.

The anti-thrombin III binding activity to the immobilized heparin wasexpressed in picomoles ATIII bound per square centimeter of substratematerial (pmol/cm²). One set of control samples was not sterilized.Another set of control samples was subjected to EtO sterilization in theabsence of a biologically compatible organic composition non-covalentlycombined with the immobilized heparin and covering material. Eachremaining bar represents the anti-thrombin III binding activity ofimmobilized heparin in the presence of the indicated biologicallycompatible organic composition non-covalently combined with theimmobilized heparin and covering material. All bars represent meanvalues of n=3 samples, except for dextran sulfate with n=6 samples.

As can be seen from the bar graph, sterilized control samples showed adramatic reduction in anti-thrombin III binding activity compared tounsterilized control samples. The anti-thrombin III binding activity ofthe unsterilized control samples was 103 pmol/cm² substrate material.The anti-thrombin III binding activity of the sterilized control sampleswas 66 pmol/cm² substrate material. EtO sterilization caused athirty-six percent (36%) reduction in anti-thrombin III binding activitycompared to the unsterilized samples.

The influence of the above described biologically compatible organiccompositions non-covalently combined with the immobilized heparin andcovering material on the anti-thrombin III binding activity followingsterilization is summarized in the following paragraph. Eachbiologically compatible organic composition was rinsed, as describedearlier, from each construction before the anti-thrombin III bindingactivity was determined.

When heparin was added to the construction, the mean anti-thrombin IIIbinding activity was 108 pmol/cm². Addition of dextran to theconstruction resulted in a mean anti-thrombin III binding activity of 98pmol/cm² substrate material. When dextran sulfate was added to theconstruction, the mean anti-thrombin III binding activity was 134pmol/cm² substrate material. Additionally, polyethylene glycol resultedin a mean anti-thrombin III binding activity of 129 pmol/cm² substratematerial. Interestingly, these values are greater than the mean valuesfor the unsterilized control samples at 103 pmol/cm² substrate material.

When inorganic calcium chloride (CaCl₂) was added to the construction,the mean anti-thrombin III binding activity of the immobilized heparinwas 75 pmol/cm² substrate material. Addition of dexamethasone to theconstruction resulted in a mean anti-thrombin III binding activity of 42pmol/cm² substrate material. DEAE dextran seemed to diminish theanti-thrombin III binding activity of the immobilized heparin with amean activity of 5 pmol/cm² substrate material.

These results demonstrate the ability to maintain, or increase, theanti-thrombin III binding activity of end point attached heparinfollowing EtO sterilization with an appropriate biologically compatiblecomposition non-covalently combined with the immobilized heparin andcovering material.

Example 3

This example describes the ability of an additional biologicallycompatible organic composition to produce a high anti-thrombin III(ATIII) binding activity of heparin end point attached to a polymericcovering material on a substrate material that is a component of animplantable medical device.

The implantable medical device used in this example was in the form of anitinol wire reinforced tube made of a porous, expanded,polytetrafluoroethylene (ePTFE) material obtained from W.L. Gore &Associates, Inc., Flagstaff, Ariz. under the tradename VIABAHN®Endoprosthesis. The tubular device was fifteen centimeters (15 cm) inlength and six millimeters (6 mm) in diameter.

The VIABAHN® Endoprosthesis was constrained within a delivery catheterand required removal from the catheter before immobilizing heparinthereon. Each catheter-constrained device was removed for processing bypulling a release cord attached to a constraining sheath and releasingthe sheath from around the device. Once unconstrained, each device wasexpanded and used as a separate substrate material. Each substratematerial (endoprosthetic device) was immersed in a PEI solution (5% inDI water) and IPA (USP grade) in a volume percent ratio of 30:70,respectively, for about twelve hours (12 hr) to place a polymericcovering material (18) on the substrate material (12). The polymericcovering material (18) had a multiplicity of reactive chemical groups(16) to which a plurality of aldehyde-modified heparin molecules (17)were eventually end point attached.

At least one additional layer of covering material (18 a, 18 b) wasplaced on the first PEI layer (18). This was performed by placing eachendoprosthetic device within a separate silicone tube and the tubeconnected to a peristaltic pump and solution-reservoir. This allowed anadditional solution containing a covering material to be repeatedlypassed through the center of the tubular medical device to coatprimarily the inside surfaces of the device.

With each endoprosthesis contained within one of these dynamic flowsystems, a covering material (18) in the form of an aqueous solution of0.10% (pH 9.0) PEI and IPA in a volume percent ratio of 45:55,respectively, was passed through the device for about twenty minutes (20min). Each device was then rinsed in DI water (pH 9.0) for five minutes(5 min) and the PEI layers cross-linked (19) by exposure to a 0.05%aqueous glutaraldehyde solution (pH 9.0) for twenty minutes (20 min).The devices were then rinsed again with an aqueous solution of PEI(0.10%, pH 9.0) for five minutes (5 min). The resultant imines werereduced with a sodium cyanborohydride solution (5 g in 1 L DI water, pH9.0) for fifteen minutes (15 min) and rinsed in DI water for thirtyminutes (30 min).

An intermediate ionic charge layer was placed on the cross-linked PEIlayer(s) of each device by flowing a solution of dextran sulfate (0.15 gdextran sulfate and one hundred grams sodium chloride (100 g NaCl)dissolved in one liter (1 L) of DI water, pH 3) through the dynamic flowsystem and over the PEI layer at sixty degrees centigrade (60° C.) forabout ninety minutes (90 min). This was followed by rinsing the systemwith DI water for fifteen minutes (15 min).

A “capping” layer (18 b) of PEI was added to the ionically chargeddextran sulfate layer (18 a) by flowing an aqueous solution of PEI(0.075%, pH 9.0) through the dynamic flow system for about forty-fiveminutes (45 min) followed by a rinse in a sodium chloride solution (50 gNaCl dissolved in 1 L DI water) for fifteen minutes (15 min). The rinsewas followed by a brief DI water flush for about two and a half minutes(2.5 min).

Aldehyde modified heparin was end point attached, or conjugated, to thePEI layer(s) by placing the construction in a heparin-containing sodiumchloride salt solution (1.5 g heparin, 29.3 g NaCl dissolved in 1 L DIwater, pH 3.9) for one hundred twenty minutes (120 min) at sixty degreescentigrade (60° C.). A 2.86 mL volume of a 2.5% (w/v) aqueous NaCNBH₃solution was added to the one liter (1 L) heparin solution ten minutes(10 min) after beginning the step. A first rinse in DI water for fifteenminutes (15 min), was followed by a rinse in a boric acid solution (0.7g NaCl, 10.6 g boric acid and 2.7 g NaOH dissolved in 1 L DI water, pH9.0) for about twenty minutes (20 min), and a final rinse in DI waterfor fifteen minutes (15 min). The construction was then subjected to alyophilization process. Staining of selected samples with toluidine blueproduced a consistent purple surface indicating uniformly bound heparin.

Based on the results obtained in the studies described in Example 2,supra, USP grade heparin (sodium salt) and 8,000 MW dextran sulfate(sodium salt) at a concentration of 0.5 g/100 ml DI water (pH9.6), werechosen as the preferred biologically compatible organic compositions tomaintain, or stabilize, the anti-thrombin III binding activity of theimmobilized heparin during and after EtO-sterilization.

For each preferred biologically compatible organic composition, sectionsof the endoprostheses having heparin end-point attached to a polymericcovering material were placed in plastic tubes containing a solution ofsaid biologically compatible organic compositions (each at aconcentration of 0.5 g/100 mL DI water, pH 9.6) and incubated at sixtydegrees centigrade (60° C.) for one hour (1 hr). Each treated sample wasremoved from the plastic tube and exposed to a lyophilization process.

Each lyophilized sample was placed in an individual Tower DUALPEEL® SelfSealing Pouch (Allegiance Healthcare Corp., McGraw Park, Ill.) andsealed for EtO sterilization. Ethylene oxide sterilization was carriedout under conditions of conditioning for one hour (1 hr), an EtO gasdwell time of one hour (1 hr), a set point temperature of fifty-fivedegrees centigrade (55° C.), and an aeration time of twelve hours (12hr).

After EtO sterilization, each construction was removed from its pouchand washed in DI water for fifteen minutes (15 min), borate buffersolution (10.6 g boric acid 2.7 g NaOH and 0.7 g NaCl dissolved in 1 LDI water, pH 9.0) for twenty minutes (20 min), and finally a rinse in DIwater for fifteen minutes (15 min).

Samples of substrate material from each EtO-sterilized device (approx.0.5 cm long) were cut from each device and the immobilized heparinmeasured for biological activity using the above-described ATIII bindingassay (Example 2). Samples were kept wet throughout the assay process.The results were expressed as picomoles of anti-thrombin III bound perarea unit of substrate material (pmol/cm²) as measured on the luminalsurface of each device and not the entire surface area of the device(i.e., both abluminal and luminal surfaces).

FIG. 10 is a bar graph illustrating the effect of two separatebiologically compatible organic compositions in the form of heparin anddextran sulfate on anti-thrombin III binding activity of heparinimmobilized on a covered substrate material during and after exposure toan EtO sterilization regimen. Anti-thrombin III binding activity isexpressed as picomoles of bound anti-thrombin III per square centimeterof substrate material. As seen from the results, the use of heparin anddextran sulfate biologically compatible organic compositions resulted inhigh anti-thrombin III binding activity to immobilized heparin followingEtO sterilization, with activities of 97 pmol/cm² substrate material and91 pmol/cm² substrate material, respectively. All bars represent meanvalues of n=6 samples.

Example 4

This example describes construction of an embodiment of the presentinvention having an aldehyde modified heparin compound end pointattached to a polymeric covering material that includes an ionicallyneutral first covering layer. The construction had heparin ATIII bindingthat was not significantly diminished by exposure to EtO sterilization.

The covering material used as a base coat in this construction waschosen to render a heparin-containing covering material, or coating,that had essentially no ionic charge. Polyvinyl alcohol and PEI wereused as the covering materials.

In accordance with U.S. Pat. No. 6,653,457, which is incorporated hereinby reference, an aldehyde modified heparin composition was bound to acovered substrate material. The substrate material (12) was expandedpolytetrafluoroethylene (ePTFE) material. An additional biocompatibleorganic chemical composition (100) was incorporated into theheparin-containing covering material (18) of the construction to enablethe heparin to undergo EtO sterilization without significant loss inbiological activity.

An ePTFE substrate material in sheet form was obtained from W.L. Gore &Associates, Inc., Flagstaff, Ariz. under the tradename GORE™Microfiltration Media (GMM-406). A layer of covering material, or basecoat, was applied to the ePTFE substrate material by mounting thematerial on a 10 cm diameter plastic embroidery hoop and immersing thesupported ePTFE material in a solution of 100% IPA for about fiveminutes (5 min). This was followed by immersion of the ePTFE material inan aqueous two percent (2%) solution of USP grade polyvinyl alcohol(PVA) (Spectrum) for fifteen minutes (15 min). After a fifteen minute(15 min) rinse in DI water, the PVA layer was exposed to a solution oftwo percent (2%) aqueous glutaraldehyde and one percent (1%)hydrochloric acid (HCL) for fifteen minutes (15 min) to cross-link (19)the PVA (18), in situ. The construction was rinsed in DI water forfifteen minutes (15 min) followed by a second fifteen minute (15 min) DIwater rinse. The resulting cross-linked PVA base coating had no netionic charge.

Another layer of polymeric covering material (18 a) was added to theconstruction by immersing the construction in an aqueous 0.15% solutionof PEI (pH 10.5) solution for thirty minutes (30 min). The resultantimines were reduced by immersing the construction in an aqueous solutionof sodium cyanborohydride solution (5 g/L in DI water, pH 10.5) forfifteen minutes (15 min). The construction was rinsed in DI water forfifteen minutes (15 min) followed by a second fifteen minute (15 min) DIwater rinse.

A covered ePTFE substrate material having a multiplicity of reactivechemical groups thereon was immersed in the heparin solution (1.0 gheparin, 29.3 g NaCl dissolved in 1 L DI water, pH 3.9) for ninetyminutes (90 min) at 60° C. A 2.86 mL volume of a 2.5% (w/v) aqueousNaCNBH₃ solution was added to the 1 L heparin solution prior tobeginning this step. A first fifteen minute (15 min) rinse in DI water,was followed by a rinse in an aqueous boric acid solution (0.7 g NaCl,10.6 g boric acid, 2.7 g NaOH dissolved in 1 L DI water, pH 9.0) forabout twenty minutes (20 min), and a final rinse in DI water rinse forfifteen minutes (15 min). The construction was then subjected to alyophilization process. Samples of the construction were then stainedwith toluidine blue. The staining produced a consistent purple surfaceindicating uniformly bound heparin on the covered ePTFE material.

The construction was exposed to an aqueous treatment solution containinga biologically compatible organic composition (100) in the form of 8,000MW USP grade dextran sulfate (sodium salt) (Sigma) by immersing theconstruction in 100 ml treatment solution (0.5 g of dextran sulfate/100mL DI water, pH 9.6) at sixty degrees centigrade (60° C.) for one hour(1 hr). Following removal of the construction from the treatmentsolution, the construction was lyophilized.

Each lyophilized construction was placed in a Tower DUALPEEL® Self SealPouch (Alligiance Healthcare Corp., McGaw Park, Ill.) for EtOsterilization. Ethylene oxide sterilization was carried out underconditions of conditioning for one hour (1 hr), an EtO gas dwell time ofone hour (1 hr), a set point temperature of fifty-five degreescentigrade (55° C.), and an aeration time of twelve hours (12 hr).

After EtO sterilization, each construction (including controls) wasremoved from its pouch and washed in DI water for fifteen minutes (15min), a borate buffer solution (10.6 g boric acid, 2.7 g NaOH, 0.7 gNaCl dissolved in 1 L DI water, pH 9.0) for twenty minutes (20 min), andfinally a rinse in DI water for fifteen minutes (15 min).

Samples of the membrane (approx. 1 cm²) with end-point attached heparinwere cut and the immobilized heparin measured for anti-thrombin IIIbinding activity using the above-described ATIII binding assay (Example2). Samples were kept wet throughout the assay process. The results wereexpressed as picomoles of anti-thrombin III bound per unit of substratesurface area (pmol/cm² substrate material).

FIG. 11 is a bar graph illustrating the effect of a biologicallycompatible organic composition in the form dextran sulfate onanti-thrombin III binding activity of end-point attached heparinimmobilized on a porous expanded polytetrafluoroethylene substratematerial and a covering material of polyvinyl alcohol and PEI, followingEtO sterilization. The biological activity of the immobilized heparinwas expressed as picomoles of anti-thrombin III bound per squarecentimeter of substrate material.

Unsterilized control samples had an anti-thrombin III binding activityof 150 pmol/cm² substrate material. The sterilized control samples hadan anti-thrombin III binding activity of 93 pmol/cm² substrate material.Ethylene oxide sterilized samples treated with dextran sulfate had ananti-thrombin III binding activity of 115 pmol/cm² substrate material.This value was greater than the control values for EtO-sterilizeddevices which were not exposed to a dextran sulfate treatment solution(i.e., 93 pmol/cm² substrate material), indicating the added dextransulfate increased the biological activity of the immobilized heparinfollowing EtO sterilization. Both of these constructions hadanti-thrombin III binding activity values that were significantly lowerthan the non-treated, non-EtO-sterilized, controls (150 pmol/cm²substrate material).

As seen from the results, dextran sulfate significantly impacted theanti-thrombin III binding activity of the immobilized heparin attachedto a construction with a polymeric covering material that includes anionically neutral first covering layer, following EtO sterilization. Allbars represent mean values of n=3 samples.

Example 5

This example describes the ability of an additional biologicallycompatible organic composition to maintain or increase the biologicalactivity of biologically active heparin immobilized to a coveredsubstrate material during and after imposition of a mechanical stress ofsufficient magnitude to otherwise significantly reduce the biologicalactivity of the entity.

In this example, implantable medical devices in the form of endoluminalprostheses were provided with a heparin-containing coating as describedin Example 3, supra. Each prosthesis was in the form of a nitinol wirereinforced tube made of a porous, expanded, polytetrafluoroethylene(ePTFE) material obtained from W.L. Gore & Associates, Inc., Flagstaff,Ariz. under the tradename VIABAHN® Endoprosthesis. The tubular devicewas fifteen centimeters (15 cm) in length and six millimeters (6 mm) indiameter. The same process was utilized as detailed in Example 3 forforming a heparin-containing coating on the device.

For treatment with the biologically compatible organic composition(100), substrate material (12) of the endoluminal device was preparedwith a polymeric covering material (18) having aldehyde modified heparin(17) end point attached to at least a portion thereof. Sections of theprepared device were placed in plastic tubes and incubated with aglycerol solution (5 mL Sigma-Aldrich SigmaUltra glycerol in 100 mL ofDI water, pH 9.6) at sixty degrees centigrade (60° C.) for one hour (1hr). Each treated device was removed from the plastic tube and exposedto a lyophilization process.

Each cylindrical endoprosthesis was placed over an intravasculardelivery system and mechanically compressed until it was sufficientlycompacted on the delivery system to be restrained with a constrainingsheath. Devices made according to Example 3 can withstand the mechanicalstresses associated with compaction of the endoprosthesis on thedelivery system without significant loss in the activity of the heparinincorporated in the coating.

Glycerol was chosen as the non-covalently bound biologically compatibleorganic composition (100) to maintain the biological activity of the endpoint attached heparin (17) during diametrical compaction and expansionof each test endoprosthesis. Each control endoprosthesis device sectiondid not have the non-covalently bound biologically compatible glycerolcomposition (100) included with the end point attached (i.e., covalentlybound) heparin (17) and polymeric covering material (18). Each devicewas subjected to a lyophilization process.

To compress and compact the endoluminal devices on a delivery system,each endoprosthesis was pulled through a tapered funnel with a fixeddiameter. Each endoprosthesis had six (6) sutures (Gore-Tex® CV-0, 0N05)sewn through one end to pull the devices through the funnel. Each devicewas pulled through the opening of a twenty-five milliliter (25 ml) pipettip (Falcon®, product #357525) with a diameter of about threemillimeters (3 mm) and into a glass tube with a diameter of about 3.1 mmto hold it in the compacted state.

After compaction, each endoprosthesis was deployed in a 0.9% aqueoussaline solution at thirty-seven degree centigrade (37° C.), rinsed andtested for anti-thrombin III binding activity as described herein. Theresults are shown in FIG. 12. Each endoprosthesis was prepared fortesting by washing in DI water for fifteen minutes (15 min), followed bya rinse in borate buffer solution (10.6 g boric acid 2.7 g NaOH, 0.7 gNaCl, dissolved in 1 L of DI water, pH 9.0) for twenty minutes (20 min)and a final fifteen minute (15 min) DI water rinse.

Samples of heparin-containing material from each endoprosthesis (approx.0.5 cm long) were cut and the bound heparin measured for biologicalactivity using the above-described anti-thrombin III (ATIII) bindingassay (Example 2). Samples were kept wet throughout the assay process.The results were expressed as anti-thrombin III binding per unit ofsubstrate surface area (pmol/cm² substrate material).

FIG. 12 is a bar graph illustrating the effect of a glycerol compositionwith immobilized heparin on a covered substrate material followingcompaction and expansion. Results show that the addition of glycerol toimmobilized heparin significantly improves the anti-thrombin III bindingactivity of the bound heparin following compaction and expansion of theimmobilized heparin compared to similarly treated control samples nothaving the added glycerol. All vertical bars represent mean values ofn=3 samples.

Heparin-immobilized to a polymeric covering material that did notreceive the additional glycerol biologically compatible organiccomposition, and was diametrically compacted and expanded, showed asignificant reduction in anti-thrombin III binding activity (85pmol/cm²) compared to similarly constructed and treated controlmaterials not diametrically compacted and expanded (137 pmol/cm²). Whenheparin-immobilized covered substrate materials were treated with abiologically compatible organic glycerol composition and exposed to thesame mechanical manipulations as the untreated construction, theanti-thrombin III binding activity of the immobilized heparin remainedsimilar to the control materials (129 pmol/cm²).

Example 6

This example describes the effect of the addition of a biologicallycompatible organic composition on the ATIII binding activity of thecoated medical device described in Examples 3 and 5, subjected tocompaction, expansion and EtO sterilization.

The implantable medical device used in this example was constructed inthe same way as described in Example 3. The device was in the form of anitinol wire reinforced tube made of a porous, expanded,polytetrafluoroethylene (ePTFE) material obtained from W.L. Gore &Associates, Inc., Flagstaff, Ariz. under the tradename VIABAHN®Endoprosthesis. The tubular device was fifteen centimeters (15 cm) inlength and six millimeters (6 mm) in diameter. The same process wasutilized as detailed in Examples 3 for forming a heparin-containingcoating on the device.

For treatment with the biologically compatible organic composition(100), substrate material (12) of the endoluminal device was preparedwith a polymeric covering material (18) having aldehyde modified heparin(17) end point attached to at least a portion thereof. The prepareddevice was placed in a plastic tube and incubated with a heparin andglycerol solution (0.5 g USP heparin and 5 mL glycerol dissolved in 100mL of DI water, pH 9.6) at sixty degrees centigrade (60° C.) for onehour (1 hr). The choice of these compounds is a result of the outcome ofExamples 2, 3 and 5. Each treated device was removed from the heparinand glycerol solution and exposed to a lyophilization process. Furtherprocessing and analysis of devices was identical to Example 5, supra.

FIG. 13 is a bar graph illustrating the ability of a biologicallycompatible organic composition in the form of glycerol and heparin tomaintain the biological activity of heparin immobilized to a polymericcovering material on a substrate material both during and after exposureto an EtO sterilization regimen and mechanic manipulation in the form ofcompaction and expansion of the substrate and polymeric coveringmaterial to which the heparin was immobilized. All vertical barsrepresent mean values of n=3 samples.

Heparin-immobilized covered substrate materials that did not receive theadditional glycerol and heparin biologically compatible organiccompositions and were exposed to EtO sterilization and diametricallycompacted and expanded showed a significant reduction in anti-thrombinIII binding activity (63 pmol/cm²) compared to similarly constructed andtreated control materials not subjected to EtO sterilization anddiametrical compaction and expansion (158 pmol/cm²). Whenheparin-immobilized covered substrate materials were treated with abiologically compatible organic glycerol and heparin composition andexposed to the same EtO sterilization conditions and mechanicalmanipulations as the untreated construction, the anti-thrombin IIIbinding activity of the immobilized heparin remained similar to thecontrol materials (147 pmol/cm²).

Example 7

This example demonstrates a relatively low anti-thrombin III bindingactivity of a commercially available heparin-coated medical device. Thedevice was a fifty centimeter (50 cm) long, six millimeter (6 mm)diameter, sterilized, and packaged heparin-coated vascular graftavailable under the tradename FLOWLINE BIPORE® Heparin Coated VascularGraft (Catalog Number 15TW5006N) from JOTEC GmbH (Hechingen, Germany).According to the manufacturer, the tubular vascular graft is made of anexpanded polytetrafluoroethylene (ePTFE) material with heparincovalently and ionically attached to the luminal surface of the graft.The manufacturer states that the heparin is stably and permanentlyattached to the ePTFE. Surfaces of the heparin-containing graft are saidto be anti-thrombotic.

Samples (0.5 cm long) of the heparin-containing vascular graft wereobtained and tested as described Example 2, supra. As with the inventivematerials, the anti-thrombin III binding activity of the vascular graftwere expressed as picomoles anti-thrombin III binding activity persquare centimeter of substrate material (pmol/cm²). As in previousexamples, only the luminal surface area of each device was measured, notthe entire surface area of the device. The results of the ATIII bindingassay showed that there was no anti-thrombin III binding activitydespite the claims by the manufacturer that biologically active heparinwas present on luminal surface of the vascular graft. It should be notedthat the anti-thrombin III binding activity assay is capable ofdetecting anti-thrombin III binding activity at a level of approximatelyfive picomoles per square centimeter substrate material (5 pmol/cm²substrate material) and above.

Example 8

This example describes the use of a peptide antibiotic agent as abiologically compatible organic composition in conjunction withbiologically active heparin immobilized to a covered, or coated,substrate material. The construction exhibited significant ATIII bindingafter exposure to EtO sterilization.

In this example, an ePTFE material in sheet form was obtained from W.L.Gore & Associates, Inc., Flagstaff, Ariz. under the tradename GORE™Microfiltration Media (GMM-406) and provided with a heparin-containingcoating using a process substantially equivalent to Example 2.

The above-described construction was exposed to a solution of bacitracin(72,000 units/gram) at a concentration of 0.5 g per 100 ml deionizedwater (DI water) by immersing the construction in one hundredmilliliters (100 ml) of the bacitracin solution for three hours (3 hr)at room temperature. The construction was removed from the solution andlyophilized prior to exposure to a sterilization procedure.

In preparation for EtO sterilization, each lyophilized construction wasplaced and sealed in a Tower DUALPEEL® Self-Seal Pouch (AllegianceHealthcare Corp., McGaw Park, Ill.). Ethylene oxide sterilization wascarried out under conditions of conditioning for one hour (1 hr), an EtOgas dwell time of one hour (1 hr), a set point temperature of fifty-fivedegree centigrade (55° C.), and an aeration time of twelve hours (12hr).

After EtO sterilization, the construction was removed from its pouch andwashed in DI water for fifteen minutes (15 min), borate buffer solution(10.6 g boric acid, 2.7 g NaOH and 0.7 g NaCl dissolved in 1000 ml of DIwater, pH 9.0) for twenty minutes (20 min), and finally rinsed in DIwater for fifteen minutes (15 min).

Samples of the membrane (approx. 1 cm²) with end-point attached heparinwere cut from the sterilized construction and the immobilized heparinmeasured for anti-thrombin III binding activity using theabove-described ATIII binding assay (Example 2). Samples were kept wetthroughout the assay process. The results were expressed as picomoles ofanti-thrombin III bound per unit of substrate surface area (pmol/cm²).

The sample treated with bacitracin and subsequently sterilized withethylene oxide had an anti-thrombin III binding activity of 9 pmol/cm²(n=3).

Example 9

This example describes the addition of a biologically compatible organiccomposition to biologically active heparin immobilized to a covered, orcoated, substrate material and previously exposed to EtO sterilization.A peptide antibiotic agent was selected as the biologically compatibleorganic composition in this example. A construction treated in this wayhad significant heparin ATIII binding after exposure to EtOsterilization.

In this example, an ePTFE material in sheet form was obtained from W.L.Gore & Associates, Inc., Flagstaff, Ariz. under the tradename GORE™Microfiltration Media (GMM-406) and provided with a heparin-containingcoating using a process substantially equivalent to Example 2.

In preparation for EtO sterilization, each lyophilized construction wasplaced and sealed in a Tower DUALPEEL® Self-Seal Pouch (AllegianceHealthcare Corp., McGaw Park, Ill.). Ethylene oxide sterilization wascarried out under conditions of conditioning for one hour (1 hr), an EtOgas dwell time of one hour (1 hr), a set point temperature of fifty-fivedegree centigrade (55° C.), and an aeration time of twelve hours (12hr).

After EtO sterilization, the construction was aseptically handled in aNUAIRE Biological Safety Cabinets, class II, type A/B3, model NU-425-600(Plymouth, Minn.).

Sterilized samples approximately one square centimeter (1 cm²) in sizewere cut from the construction and submerged in a filter-sterilizedbacitracin solution (649.4 mg at 77000 units/g dissolved in 10 ml of0.9% sodium chloride irrigation solution purchased from Hospira, Inc.)with a resultant concentration of approximately five thousand (5000)units per ml of USP grade 0.9% sodium chloride irrigation solution.Samples were exposed to the bacitracin solution for two minutes (2 min)at room temperature.

Samples were removed from the solution and washed in DI water forfifteen minutes (15 min), borate buffer solution (10.6 g boric acid, 2.7g NaOH and 0.7 g NaCl dissolved in 1000 ml of DI water, pH 9.0) fortwenty minutes (20 min), and finally a rinse in DI water for fifteenminutes (15 min).

Samples of the sheet material (approx. 1 cm²) with end-point attachedheparin were cut and the immobilized heparin measured for anti-thrombinIII binding activity using the above-described ATIII binding assay(Example 2). Samples were kept wet throughout the assay process. Theresults were expressed as picomoles of anti-thrombin III bound per unitof substrate surface area (pmol/cm²).

The sample that was initially sterilized with ethylene oxide andsubsequently treated with bacitracin had an anti-thrombin III bindingactivity of 185 pmol/cm² pmol/cm² (n=3). As these results indicate, atherapeutic agent can be admixed with biologically active heparinimmobilized to a covered substrate material after the entity has beensterilized without significantly reducing its biological activity.

Example 10

This example demonstrates biologically active heparin immobilized to acovered substrate material that was admixed with a biologicallycompatible organic composition, EtO sterilized, and finally treated witha peptide antibiotic agent. A construction treated in this way hassignificant heparin ATIII binding.

In this example, an ePTFE material in sheet form was obtained from W.L.Gore & Associates, Inc., Flagstaff, Ariz. under the tradename GORE™Microfiltration Media (GMM-406) and provided with a heparin-containingcoating using a process substantially equivalent to Example 2.

The above-described construction was exposed to a solution ofpolyethylene glycol (20,000 molecular weight, Sigma) at a concentrationof 0.5 g per 100 ml DI water adjusted to pH 9.6. The construction wasplaced into a beaker and one hundred milliliters (100 ml) was added tocompletely immerse the construction in the polyethylene glycol solution.The construction was exposed to the polyethylene glycol solution for onehour (1 hr) at sixty degrees centigrade (60° C.). The construction wasremoved from the solution and lyophilized prior to exposure to asterilization procedure.

In preparation for EtO sterilization, the lyophilized construction wasplaced and sealed in a Convertors® Self-Seal Pouch (Cardinal Health,McGaw Park, Ill.). Ethylene oxide sterilization was carried out underconditions of conditioning for one hour (1 hr), an EtO gas dwell time ofone hour (1 hr), a set point temperature of fifty-five degree centigrade(55° C.), and an aeration time of twelve hours (12 hr).

After EtO sterilization, the construction was aseptically handled in aNUAIRE Biological Safety Cabinet, class II, type A/B3, model NU-425-600(Plymouth, Minn.).

Sterilized samples approximately one square centimeter (1 cm²) in sizewere cut from the construction and submerged in a filter-sterilizedbacitracin solution (649.4 mg at 77,000 units/g dissolved in 10 ml of0.9% sodium chloride irrigation solution) with a resultant concentrationof approximately five thousand (5,000) units per ml of USP grade 0.9%sodium chloride irrigation solution. Samples were exposed to thebacitracin solution for two minutes (2 min) at room temperature.

Samples were removed from the solution and washed in DI water forfifteen minutes (15 min), borate buffer solution (10.6 g boric acid, 2.7g NaOH and 0.7 g NaCl dissolved in 1,000 ml of DI water, pH 9.0) fortwenty minutes (20 min), and finally a rinse in DI water for fifteenminutes (15 min).

Samples of the membrane (approx. 1 cm²) with end-point attached heparinwere cut and the immobilized heparin measured for anti-thrombin IIIbinding activity using the above-described ATIII binding assay (Example2). Samples were kept wet throughout the assay process. The results wereexpressed as picomoles of anti-thrombin III bound per unit of substratesurface area (pmol/cm²).

The sample that was initially treated with polyethylene glycol,sterilized with ethylene oxide, and then treated with bacitracin had ananti-thrombin III binding activity of 195 pmol/cm² (n=3). Hence, asterilized covered substrate material with a biologically active heparinimmobilized thereto and a first biologically compatible organiccomposition (PEG) admixed therewith can be further treated with a secondbiologically compatible organic composition (bacitracin) following EtOsterilization and retain significant ATIII binding activity.

Example 11

This example demonstrates the covalent attachment of biologically activeheparin to a covering material, or coating layer, placed on an expandedpolytetrafluoroethylene (ePTFE) material followed by the subsequentcovalent attachment of a biologically compatible organic composition(aldehyde activated dextran) to the covering material. This compositionwas exposed to EtO sterilization and thereafter demonstrated significantbiological heparin activity.

In this example, an ePTFE material in sheet form was obtained from W.L.Gore & Associates, Inc., Flagstaff, Ariz. under the tradename GORE™Microfiltration Media (GMM-406). This ePTFE material was provided with aheparin-containing coating using a process substantially equivalent toExample 2, however, the construction was stored in DI water after beingcoated rather than lyophilized.

The above-described construction coated with a covering material wasexposed to an aldehyde-activated dextran (40,000 molecular weight,Pierce) solution (0.050 g aldehyde-activated dextran, 2.93 g NaCldissolved in 100 ml DI water, pH 5.5) for one hundred twenty minutes(120 min) at sixty degrees centigrade (60° C.). A 0.286 mL volume of a2.5% (w/v) aqueous NaCNBH₃ solution was added to the one hundredmilliliters (100 ml) aldehyde-activated dextran solution prior to addingthe sample.

The construction was removed from the aldehyde-activated dextransolution and washed in DI water for fifteen minutes (15 min), boratebuffer solution (10.6 g boric acid, 2.7 g NaOH and 0.7 g NaCl dissolvedin 1,000 ml of DI water, pH 9.0) for twenty minutes (20 min), andfinally a rinse in DI water for fifteen minutes (15 min) followed bylyophilization of the entire construction to produce dry heparin boundto the ePTFE material.

In preparation for EtO sterilization, the lyophilized construction wasplaced and sealed in a Convertors® Self-Seal Pouch (Cardinal Health,McGaw Park, Ill.). Ethylene oxide sterilization was carried out underconditions of conditioning for one hour (1 hr), an EtO gas dwell time ofone hour (1 hr), a set point temperature of fifty-five degree centigrade(55° C.), and an aeration time of twelve hours (12 hr).

Samples of the sterilized membrane (approx. 1 cm²) with end-pointattached heparin were cut and the immobilized heparin measured foranti-thrombin III binding activity using the above-described ATIIIbinding assay (Example 2). The results were expressed as picomoles ofanti-thrombin III bound per unit of substrate surface area (pmol/cm²).

The sample prepared as described in this example had a meananti-thrombin III binding activity of 65 pmol/cm² (n=3). This exampledemonstrates that a biocompatible organic composition can, in additionto the covalently bound end-point attached heparin, be covalentlyattached to the coating layer while maintaining significant heparinactivity following EtO sterilization.

Example 12

This example demonstrates the covalent attachment of biologically activeheparin to a covering material, or coating layer, placed on an expandedpolytetrafluoroethylene (ePTFE) material followed by the subsequentcovalent attachment of a biologically compatible organic composition(aldehyde activated polyethylene glycol, 1,000 molecular weight) to thecovering material. This composition was exposed to EtO sterilization andthereafter demonstrated significant biological heparin activity.

In this example, an ePTFE material in sheet form was obtained from W.L.Gore & Associates, Inc., Flagstaff, Ariz. under the tradename GORE™Microfiltration Media (GMM-406). This ePTFE material was provided with aheparin-containing coating using a process substantially equivalent toExample 2, however, the construction was stored in DI water after beingcoated rather than lyophilized.

The above-described construction coated with a covering material wasexposed to an aldehyde activated PEG (1,000 molecular weight, Nanocs)solution (0.20 g PEG, 3.90 g NaCl dissolved in 133 ml DI water, pH 5.5)for one hundred twenty minutes (120 min) at sixty degrees centigrade(60° C.). A 0.380 mL volume of a 2.5% (w/v) aqueous NaCNBH₃ solution wasadded to the one hundred milliliters (100 ml) PEG solution prior toadding the sample.

The construction was removed from the PEG solution and washed in DIwater for fifteen minutes (15 min), borate buffer solution (10.6 g boricacid, 2.7 g NaOH and 0.7 g NaCl dissolved in 1,000 ml of DI water, pH9.0) for twenty minutes (20 min), and finally a rinse in DI water forfifteen minutes (15 min) followed by lyophilization of the entireconstruction to produce dry heparin bound to the ePTFE material.

In preparation for EtO sterilization, the lyophilized construction wasplaced and sealed in a Convertors® Self-Seal Pouch (Cardinal Health,McGaw Park, Ill.). Ethylene oxide sterilization was carried out underconditions of conditioning for one hour (1 hr), an EtO gas dwell time ofone hour (1 hr), a set point temperature of fifty-five degree centigrade(55° C.), and an aeration time of twelve hours (12 hr).

Samples of the sterilized membrane (approx. 1 cm²) with end-pointattached heparin were cut and the immobilized heparin measured foranti-thrombin III binding activity using the above-described ATIIIbinding assay (Example 2). The results were expressed as picomoles ofanti-thrombin III bound per unit of substrate surface area (pmol/cm²).

The sample prepared as described in this example had a meananti-thrombin III binding activity of 96 pmol/cm² (n=3). This exampledemonstrates a biocompatible organic composition can, in addition to thecovalently bound end-point attached heparin, be covalently attached tothe coating layer while maintaining significant heparin activity.

Example 13

This example demonstrates the covalent attachment of biologically activeheparin to a covering material, or coating layer, placed on an expandedpolytetrafluoroethylene (ePTFE) material followed by the subsequentcovalent attachment of a biologically compatible organic composition(aldehyde activated polyethylene glycol, 5,000 molecular weight) to thecovering, or coating, material. This composition was exposed to EtOsterilization and thereafter demonstrated significant biological heparinactivity.

In this example, an ePTFE material in sheet form was obtained from W.L.Gore & Associates, Inc., Flagstaff, Ariz. under the tradename GORE™Microfiltration Media (GMM-406). This ePTFE material was provided with aheparin-containing coating using a process substantially equivalent toExample 2, however, the construction was stored in DI water after beingcoated rather than lyophilized.

The above-described construction coated with a covering material wasexposed to an aldehyde activated PEG (5,000 molecular weight, Nanocs)solution (0.20 g PEG, 3.90 g NaCl dissolved in 133 ml DI water, pH 5.5)for one hundred twenty minutes (120 min) at sixty degrees centigrade(60° C.). A 0.380 mL volume of a 2.5% (w/v) aqueous NaCNBH₃ solution wasadded to the one hundred milliliters (100 ml) PEG solution prior toadding the sample.

The construction was removed from the PEG solution and washed in DIwater for fifteen minutes (15 min), borate buffer solution (10.6 g boricacid, 2.7 g NaOH and 0.7 g NaCl dissolved in 1,000 ml of DI water, pH9.0) for twenty minutes (20 min), and finally a rinse in DI water forfifteen minutes (15 min) followed by lyophilization of the entireconstruction to produce dry heparin bound to the ePTFE material.

In preparation for EtO sterilization, the lyophilized construction wasplaced and sealed in a Convertors® Self-Seal Pouch (Cardinal Health,McGaw Park, Ill.). Ethylene oxide sterilization was carried out underconditions of conditioning for one hour (1 hr), an EtO gas dwell time ofone hour (1 hr), a set point temperature of fifty-five degree centigrade(55° C.), and an aeration time of twelve hours (12 hr).

Samples of the sterilized membrane (approx. 1 cm²) with end-pointattached heparin were cut and the immobilized heparin measured foranti-thrombin III binding activity using the above-described ATIIIbinding assay (Example 2). The results were expressed as picomoles ofanti-thrombin III bound per unit of substrate surface area (pmol/cm²).

The sample prepared as described in this example had a meananti-thrombin III binding activity of 64 pmol/cm² (n=3). This exampledemonstrates that a biocompatible organic composition can, in additionto the covalently bound end-point attached heparin, be covalentlyattached to the coating layer while maintaining significant heparinactivity.

Example 14

This example demonstrates the covalent attachment of biologically activeheparin to a covering material, or coating layer, placed on an expandedpolytetrafluoroethylene (ePTFE) material followed by the subsequentcovalent attachment of a biologically compatible organic composition(EDC activated USP heparin) to the covering material. This compositionwas exposed to EtO sterilization and thereafter demonstrated significantbiological heparin activity.

In this example, an ePTFE material in sheet form was obtained from W.L.Gore & Associates, Inc., Flagstaff, Ariz. under the tradename GORE™Microfiltration Media (GMM-406). This ePTFE material was provided with aheparin-containing coating using a process substantially equivalent toExample 2, however, the construction was stored in DI water after beingcoated rather than lyophilized.

USP grade heparin was attached, or conjugated, to the PEI layer(s)already containing end point attached heparin by placing theconstruction in a USP grade heparin-containing sodium chloride saltsolution (1.5 g USP heparin, 29.3 g NaCl dissolved in 1 L DI water, pH3.9) for one hundred twenty minutes (120 min) at sixty degreescentigrade (60° C.). The constructions were transferred to a solution of0.1 M MES [2-(N-morpholino)ethanesulfonic acid] BupH™ MES bufferedsaline (Pierce), 1.5 g USP heparin, 29.3 g NaCl, 0.20 gN-(3-Dimethylaminopropyl)-W-ethylcarbodiimide hydrochloride (EDC), and0.13 g N-hydroxysulfosuccinimide (NHS) dissolved in 1 L DI water, at pH5.5 for 4 hours (4 hr) at room temperature.

The construction was removed from the above-described solution andwashed in DI water for fifteen minutes (15 min), borate buffer solution(10.6 g boric acid, 2.7 g NaOH and 0.7 g NaCl dissolved in 1000 ml of DIwater, pH 9.0) for twenty minutes (20 min), and finally a rinse in DIwater for fifteen minutes (15 min) followed by lyophilization of theentire construction to produce dry heparin bound to the ePTFE material.

In preparation for EtO sterilization, the lyophilized construction wasplaced and sealed in a Convertors® Self-Seal Pouch (Cardinal Health,McGaw Park, Ill.). Ethylene oxide sterilization was carried out underconditions of conditioning for one hour (1 hr), an EtO gas dwell time ofone hour (1 hr), a set point temperature of fifty-five degree centigrade(55° C.), and an aeration time of twelve hours (12 hr).

Samples of the sterilized membrane (approx. 1 cm²) with end-pointattached heparin were cut and the immobilized heparin measured foranti-thrombin III binding activity using the above-described ATIIIbinding assay (Example 2). The results were expressed as picomoles ofanti-thrombin III bound per unit of substrate surface area (pmol/cm²).

The sample prepared as described in this example had a meananti-thrombin III binding activity of 31 pmol/cm² (n=3). This exampledemonstrates that a biocompatible organic composition can, in additionto the covalently bound end-point attached heparin, be covalentlyattached to the coating layer while maintaining significant heparinactivity.

Example 15

This example demonstrates covalent attachment of biologically activeheparin to a covering material, or coating layer, placed on an expandedpolytetrafluoroethylene (ePTFE) material followed by a secondarycovalent attachment of the heparin to the coating layer. To achieve thesecondary covalent attachment of the end-point attached heparin, thecarboxylic acid groups are activated with EDC and reacted with theremaining primary amine groups present in the coating layer. Thiscomposition was exposed to EtO sterilization and thereafter demonstratedsignificant biological heparin activity.

In this example, an ePTFE material in sheet form was obtained from W.L.Gore & Associates, Inc., Flagstaff, Ariz. under the tradename GORE™Microfiltration Media (GMM-406). This ePTFE material was provided with aheparin-containing coating using a process substantially equivalent toExample 2, however, the construction was stored in DI water after beingcoated rather than lyophilized.

Membranes were transferred to a solution of 0.1 M MES[2-(N-morpholino)ethanesulfonic acid] BupH™ MES buffered saline(Pierce), 29.3 g NaCl, 0.20 gN-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), and0.13 g N-hydroxysulfosuccinimide (NHS) dissolved in 1 L DI water, at pH5.5 for 4 hours (4 hr) at room temperature.

The construction was removed from the above-described solution andwashed in DI water for fifteen minutes (15 min), borate buffer solution(10.6 g boric acid, 2.7 g NaOH and 0.7 g NaCl dissolved in 1,000 ml ofDI water, pH 9.0) for twenty minutes (20 min), and finally a rinse in DIwater for fifteen minutes (15 min) followed by lyophilization of theentire construction to produce dry heparin bound to the ePTFE material.

In preparation for EtO sterilization, the lyophilized construction wasplaced and sealed in a Convertors® Self-Seal Pouch (Cardinal Health,McGaw Park, Ill.). Ethylene oxide sterilization was carried out underconditions of conditioning for one hour (1 hr), an EtO gas dwell time ofone hour (1 hr), a set point temperature of fifty-five degree centigrade(55° C.), and an aeration time of twelve hours (12 hr).

Samples of the sterilized membrane (approx. 1 cm²) with end-pointattached heparin were cut and the immobilized heparin measured foranti-thrombin III binding activity using the above-described ATIIIbinding assay (Example 2). The results were expressed as picomoles ofanti-thrombin III bound per unit of substrate surface area (pmol/cm²).

The sample prepared as described in this example had a meananti-thrombin III binding activity of 20 pmol/cm² (n=3). This exampledemonstrates that heparin can be further covalently attached to acoating layer, in addition to the covalent end-point attachment, whilemaintaining significant heparin activity following EtO sterilization.

Example 16

This example describes use of a peptide antibiotic agent as abiologically compatible organic composition in conjunction withbiologically active heparin that is immobilized to a covered substratematerial. The construction had significant ATIII binding activity aftermechanical compaction and expansion.

In this example, implantable medical devices in the form of endoluminalprostheses were heparinized using a process substantially equivalent toExample 3. Bacitracin was then applied using the conditionssubstantially equivalent to those described in Example 8. Theheparinized endoluminal prostheses were then mechanically compacted,mechanically expanded, rinsed, cut for testing, and assayed for ATIIIbinding in a manner substantially equivalent to that described inExample 5.

The sample treated with bacitracin and subsequently compacted andexpanded had a mean anti-thrombin III binding activity of 234 pmol/cm²(n=3).

Example 17

This example describes the addition of a biologically compatible organiccomposition to biologically active heparin that is immobilized to acovered substrate material and previously compacted and expanded. Apeptide antibiotic agent was selected as the biologically compatibleorganic composition. The construction treated in this way hadsignificant heparin ATIII binding after compaction and expansion.

In this example, implantable medical devices in the form of endoluminalprostheses were heparinized using a process substantially equivalent toExample 3 and mechanically compacted and expanded in a mannersubstantially equivalent to that described in Example 5. The endoluminalprosthesis was then treated with bacitracin and rinsed in a mannersubstantially equivalent to that described in Example 9. The heparinizedendoluminal prostheses were then cut for testing and assayed for ATIIIbinding as described in Example 5.

The heparinized sample that was compacted and expanded, followed by theaddition of bacitracin, had a mean anti-thrombin III binding activity of207 pmol/cm² (n=3).

Example 18

This example describes biologically active heparin immobilized to acovered substrate material. The immobilized biologically active heparinwas admixed with a biologically compatible organic composition,mechanically compacted, mechanically expanded, and treated with apeptide antibiotic agent. The construction treated in this way hadsignificant heparin ATIII binding after exposure to mechanicalmanipulation.

Endoluminal prosthesis were treated and tested as described in Example17 with one exception; polyethylene glycol was admixed with theheparinized endoluminal prosthesis, as described in Example 10, prior tocompaction and expansion in a manner substantially equivalent to that ofExample 5.

A heparinized sample with an admixed biologically compatible organiccomposition that was compacted and expanded, followed by the addition ofbacitracin, had a mean anti-thrombin III binding activity of 215pmol/cm² (n=3).

Example 19

This example demonstrates covalent attachment of biologically activeheparin to a covering material, or coating layer, placed on an expandedpolytetrafluoroethylene (ePTFE) material followed by covalent attachmentof a biologically compatible organic composition (aldehyde activateddextran) to the covering material. This composition was exposed tomechanical compaction and expansion and thereafter demonstratedsignificant biological heparin activity.

In this example, implantable medical devices in the form of endoluminalprostheses were heparinized using a process substantially equivalent toExample 3. Aldehyde activated dextran was immobilized to the coveringlayer in a manner substantially equivalent to that described in Example11. The heparinized endoluminal prostheses were then mechanicallycompacted, mechanically expanded, cut for testing, and assayed for ATIIIbinding in a manner substantially equivalent to that described inExample 5.

The sample prepared as described in this example had a meananti-thrombin III binding activity of 155 pmol/cm² (n=3).

Example 20

This example demonstrates the covalent attachment of biologically activeheparin to a covering material, or coating layer, placed on an expandedpolytetrafluoroethylene (ePTFE) material followed by the subsequentcovalent attachment of a biologically compatible organic composition(aldehyde activated polyethylene glycol, 1,000 molecular weight) to thecovering material. This composition was exposed to mechanical compactionand expansion and thereafter demonstrated significant biological heparinactivity.

In this example, implantable medical devices in the form of endoluminalprostheses were heparinized using a process substantially equivalent toExample 3. Aldehyde activated polyethylene glycol was immobilized to thecovering layer in a manner substantially equivalent to that described inExample 12. The heparinized endoluminal prostheses were then compacted,expanded, cut for testing, and assayed for ATIII binding in a mannersubstantially equivalent to that described in Example 5.

The sample prepared as described in this example had a meananti-thrombin III binding activity of 221 pmol/cm² (n=3).

Example 21

This example demonstrates the covalent attachment of biologically activeheparin to a covering material, or coating layer, placed on an expandedpolytetrafluoroethylene (ePTFE) material followed by the subsequentcovalent attachment of a biologically compatible organic composition(aldehyde activated polyethylene glycol, 5,000 molecular weight) to thecovering material. This composition was exposed to mechanical compactionand expansion and thereafter demonstrated significant biological heparinactivity.

In this example, implantable medical devices in the form of endoluminalprostheses were heparinized using a process substantially equivalent toExample 3. Aldehyde activated polyethylene glycol was immobilized to thecovering layer in a manner substantially equivalent to that described inExample 13. The heparinized endoluminal prostheses were then compacted,expanded, cut for testing, and assayed for ATIII binding in a mannersubstantially equivalent to that described in Example 5.

The sample prepared as described in this example had a meananti-thrombin III binding activity of 210 pmol/cm² (n=3).

Example 22

This example demonstrates covalent attachment of biologically activeheparin to a covering material, or coating layer, placed on an expandedpolytetrafluoroethylene (ePTFE) material followed by the subsequentcovalent attachment of a biologically compatible organic composition(EDC activated USP heparin) to the covering material. This compositionwas exposed to mechanical compaction and expansion and thereafterdemonstrated significant biological heparin activity.

In this example, implantable medical devices in the form of endoluminalprostheses were heparinized using a process substantially equivalent toExample 3. USP Heparin was immobilized to the covering layer in a mannersubstantially equivalent to that described in Example 14. Theheparinized endoluminal prostheses were then compacted, expanded, cutfor testing, and assayed for ATIII binding in a manner substantiallyequivalent to that described in Example 5.

The sample prepared as described in this example had anti-thrombin IIIbinding activity of 155 pmol/cm² (n=3).

Example 23

This example demonstrates covalent attachment of biologically activeheparin to a covering material, or coating layer, placed on an expandedpolytetrafluoroethylene (ePTFE) material followed by a secondarycovalent attachment of the heparin to reactive groups of the coatinglayer. To achieve the secondary covalent attachment of the end-pointattached heparin, the carboxylic acid groups were activated with EDC andreacted with the remaining primary amine groups present in the coatinglayer. This composition was exposed to mechanical compaction andexpansion. The construction thereafter demonstrated significantbiological heparin activity.

In this example, implantable medical devices in the form of endoluminalprostheses were heparinized using a process substantially equivalent toExample 3. Further covalent attachment of the immobilized heparin to thecovering layer was conducted in a manner substantially equivalent tothat described in Example 15. The heparinized endoluminal prostheseswere then mechanically compacted, mechanically expanded, cut fortesting, and assayed for ATIII binding in a manner substantiallyequivalent to that described in Example 5.

The samples prepared as described in this example had a meananti-thrombin III binding activity of 140 pmol/cm² (n=3).

Example 24

This example demonstrates the covalent attachment of biologically activeheparin to a covering material, or coating layer, placed on an expandedpolytetrafluoroethylene (ePTFE) material followed by the additionalattachment of a biologically compatible organic composition via a labilebond to the covering material. The labile bond allows for local deliveryof a therapeutic compound while the stably bound heparin retainedsignificant ATIII binding activity following sterilization andmechanical compaction and expansion.

In this example, implantable medical devices in the form of endoluminalprostheses were heparinized using a process substantially equivalent toExample 3. Additional aldehyde modified heparin was end point attachedto the coating layer via a labile covalent bond by placing theconstruction in a heparin-containing sodium chloride salt solution (1.5g aldehyde modified heparin, 29.3 g NaCl dissolved in 1 L DI water, pH3.9) for one hundred twenty minutes (120 min) at sixty degreescentigrade (60° C.). It is important to note that the reducing agent,NaCNBH₃, was not added during this second conjugation of aldehydemodified heparin. The bond formed between primary amines and aldehydes,when left in the un-reduced state, is labile. The samples were thenrinsed in DI water for fifteen minutes (15 min), borate buffer solution(10.6 g boric acid, 2.7 g NaOH and 0.7 g NaCl dissolved in 1 L DI water,pH 9.0) for twenty minutes (20 min), and finally in DI water for fifteenminutes (15 min) followed by lyophilization of the entire constructionwhich produced dry heparin bound to the ePTFE material.

The heparinized endoluminal prostheses were then compacted in a mannersubstantially equivalent to that described in Example 5 and sterilizedas described in Example 3. They were then expanded, cut for testing, andassayed for ATIII binding as described in Example 5.

The samples prepared as described in this example had anti-thrombin IIIbinding activity of 31 pmol/cm² (n=3).

Example 25

This example demonstrates the covalent attachment of biologically activeheparin to a covering material, or coating layer, placed on an expandedpolytetrafluoroethylene (ePTFE) material followed by the additionalattachment of a biologically compatible organic composition via a labilebond to the covering material. The labile bond allows for local deliveryof a therapeutic compound while the stably bound heparin retainedsignificant ATIII binding activity following mechanical compaction andexpansion.

In this example, implantable medical devices in the form of endoluminalprostheses were heparinized using a process substantially equivalent toExample 3. Additional aldehyde modified heparin was end point attachedto the coating layer via a labile covalent bond by placing theconstruction in a heparin-containing sodium chloride salt solution (1.5g aldehyde modified heparin, 29.3 g NaCl dissolved in 1 L DI water, pH3.9) for one hundred twenty minutes (120 min) at sixty degreescentigrade (60° C.). It is important to note that the reducing agent,NaCNBH₃, was not added during this second conjugation of aldehydemodified heparin. The bond formed between primary amines and aldehydes,when left in the un-reduced state, is labile. The samples were thenrinsed in DI water for fifteen minutes (15 min), borate buffer solution(10.6 g boric acid, 2.7 g NaOH and 0.7 g NaCl dissolved in 1 L DI water,pH 9.0) for twenty minutes (20 min), and finally in DI water for fifteenminutes (15 min) followed by lyophilization of the entire constructionto produce dry heparin bound to the ePTFE material.

The heparinized endoluminal prostheses were then compacted, expanded,cut for testing, and assayed for ATIII binding in a manner substantiallyequivalent to that described in Example 5.

The samples prepared as described in this example had a meananti-thrombin III binding activity of 195 pmol/cm² (n=3).

Example 26

This example demonstrates the covalent attachment of biologically activeheparin to a covering material, or coating layer, placed on an expandedpolytetrafluoroethylene (ePTFE) material followed by the additionalattachment of a biologically compatible organic composition via a labilebond to the covering material. The labile bond allows for local deliveryof a therapeutic compound while the stably bound heparin retainedsignificant ATIII binding activity following EtO sterilization.

In this example, an ePTFE material in sheet form was obtained from W.L.Gore & Associates, Inc., Flagstaff, Ariz. under the tradename GORE™Microfiltration Media (GMM-406) and provided with a heparin-containingcoating using a process substantially equivalent to Example 2.Additional aldehyde modified heparin was end point attached to thecoating layer via a labile covalent bond by placing the construction ina heparin-containing sodium chloride salt solution (1.5 g aldehydemodified heparin, 29.3 g NaCl dissolved in 1 L DI water, pH 3.9) for onehundred twenty minutes (120 min) at sixty degrees centigrade (60° C.).It is important to note that the reducing agent, NaCNBH₃, was not addedduring this second conjugation of aldehyde modified heparin. The bondformed between primary amines and aldehydes, when left in the un-reducedstate, is labile. The samples were then rinsed in DI water for fifteenminutes (15 min), borate buffer solution (10.6 g boric acid, 2.7 g NaOHand 0.7 g NaCl dissolved in 1 L DI water, pH 9.0) for twenty minutes (20min), and finally in DI water for fifteen minutes (15 min) followed bylyophilization of the entire construction to produce dry heparin boundto the ePTFE material.

The heparinized material was then sterilized, cut for testing, andassayed for ATIII binding as described in Example 2.

The samples prepared as described in this example had a meananti-thrombin III binding activity of 108 pmol/cm² (n=3).

Example 27

This example demonstrates the covalent attachment of biologically activeheparin to a covering material placed on an expandedpolytetrafluoroethylene (ePTFE) material followed by the subsequentcovalent attachment of a biologically compatible organic composition(aldehyde activated polyethylene glycol) to the covering material, withthe final addition of a non-covalently admixed biologically compatibleorganic composition (Bacitracin). This composition was exposed to EtOsterilization and thereafter demonstrated significant biological heparinactivity.

In this example, an ePTFE material in sheet form was obtained from W.L.Gore & Associates, Inc., Flagstaff, Ariz. under the tradename GORE™Microfiltration Media (GMM-406) and provided with a heparin-containingcoating using a process substantially equivalent to Example 2. Aldehydeactivated polyethylene glycol (1,000 molecular weight) was covalentlyattached to the covering material in a manner substantially equivalentto that described in Example 12 and Bacitracin was then non-covalentlyadmixed with this composition as described in Example 8. The compositionwas then sterilized and sampled as described in Example 8 and theimmobilized heparin was measured for anti-thrombin III binding activityusing the ATIII binding assay described in Example 2.

The samples prepared as described in this example had a meananti-thrombin III binding activity of 126 pmol/cm² (n=3).

Example 28

This example demonstrates the covalent attachment of biologically activeheparin to a covering material placed on an expandedpolytetrafluoroethylene (ePTFE) material followed by the subsequentcovalent attachment of a biologically compatible organic composition(aldehyde activated polyethylene glycol) to the covering material, withthe final addition of a non-covalently admixed biologically compatibleorganic composition (Bacitracin). This composition was exposed tomechanical compaction and expansion and thereafter demonstratedsignificant biological heparin activity.

In this example, implantable medical devices in the form of endoluminalprostheses were heparinized using a process substantially equivalent toExample 3. Aldehyde activated polyethylene glycol (1,000 molecularweight) was covalently attached to the covering material in a mannersubstantially equivalent to that described in Example 12 and Bacitracinwas then non-covalently admixed with this composition as described inExample 8. The heparinized endoluminal prostheses were then mechanicallycompacted, mechanically expanded, cut for testing, rinsed, and assayedfor ATIII binding in a manner substantially equivalent to that describedin Example 5.

The sample prepared as described in this example had a meananti-thrombin III binding activity of 249 pmol/cm² (n=3).

Example 29

This example demonstrates the covalent attachment of biologically activeheparin to a covering material placed on an expandedpolytetrafluoroethylene (ePTFE) material followed by the subsequentcovalent attachment of a biologically compatible organic composition(aldehyde activated dextran) to the covering material, with the finaladdition of a non-covalently admixed biologically compatible organiccomposition (dexamethasone). This composition was exposed to EtOsterilization and thereafter demonstrated significant biological heparinactivity.

In this example, an ePTFE material in sheet form was obtained from W.L.Gore & Associates, Inc., Flagstaff, Ariz. under the tradename GORE™Microfiltration Media (GMM-406) and provided with a heparin-containingcoating using a process substantially equivalent to Example 2. Aldehydeactivated dextran was covalently attached to the covering material in amanner substantially equivalent to that described in Example 11 anddexamethasone was then non-covalently admixed with this compositionwhich was then sterilized and sampled as described in Example 2. Theimmobilized heparin was measured for anti-thrombin III binding activityusing the ATIII binding assay described in Example 2.

The samples prepared as described in this example had a meananti-thrombin III binding activity of 77 pmol/cm² (n=3).

Example 30

This example demonstrates the covalent attachment of biologically activeheparin to a covering material placed on an expandedpolytetrafluoroethylene (ePTFE) material followed by the subsequentcovalent attachment of a biologically compatible organic composition(aldehyde activated dextran) to the covering material, with the finaladdition of a non-covalently admixed biologically compatible organiccomposition (dexamethasone). This composition was exposed to mechanicalcompaction and expansion and thereafter demonstrated significantbiological heparin activity.

In this example, implantable medical devices in the form of endoluminalprostheses were heparinized using a process substantially equivalent toExample 3. Aldehyde activated dextran was covalently attached to thecovering material as described in a manner substantially equivalent tothat Example 11 and dexamethasone was then non-covalently admixed withthis composition as described in Example 2. The heparinized endoluminalprostheses were then mechanically compacted, mechanically expanded, cutfor testing, rinsed, and assayed for ATIII binding in a mannersubstantially equivalent to that described in Example 5.

The sample prepared as described in this example had a meananti-thrombin III binding activity of 197 pmol/cm² (n=3).

Example 31

This example demonstrates the covalent attachment of biologically activeheparin to a covering material, or coating layer, placed on an expandedpolytetrafluoroethylene (ePTFE) material followed by the additionalattachment of a biologically compatible organic composition(polyethylene glycol) via a labile bond to the covering material, withthe final addition of a non-covalently admixed biologically compatibleorganic composition (dexamethasone). This composition was exposed to EtOsterilization and thereafter demonstrated significant biological heparinactivity.

In this example, an ePTFE material in sheet form was obtained from W.L.Gore & Associates, Inc., Flagstaff, Ariz. under the tradename GORE™Microfiltration Media (GMM-406) and provided with a heparin-containingcoating using a process substantially equivalent to Example 2. Aldehydemodified polyethylene glycol (1,000 MW) was attached to the coatinglayer via a labile covalent bond using a process substantiallyequivalent to that described in Example 12 excluding the addition ofNaCNBH₃. It is important to note that the reducing agent, NaCNBH₃, wasnot added during the conjugation of aldehyde modified polyethyleneglycol. The bond formed between primary amines and aldehydes, when leftin the un-reduced state, is labile. The samples were then rinsed in DIwater for fifteen minutes (15 min), borate buffer solution (10.6 g boricacid, 2.7 g NaOH and 0.7 g NaCl dissolved in 1 L DI water, pH 9.0) fortwenty minutes (20 min), and finally in DI water for fifteen minutes (15min) followed by lyophilization of the entire construction to producedry heparin and polyethylene glycol bound to the ePTFE material.Dexamethasone was then non-covalently admixed with this composition asdescribed in Example 2. The heparinized material was then sterilized,sampled, and measured for anti-thrombin III binding activity using theATIII binding assay described in Example 2.

The samples prepared as described in this example had a meananti-thrombin III binding activity of 114 pmol/cm² (n=3).

Example 32

This example demonstrates the covalent attachment of biologically activeheparin to a covering material, or coating layer, placed on an expandedpolytetrafluoroethylene (ePTFE) material followed by the additionalattachment of a biologically compatible organic composition(polyethylene glycol) via a labile bond to the covering material, withthe final addition of a non-covalently admixed biologically compatibleorganic composition (dexamethasone). This composition was exposed tomechanical compaction and expansion and thereafter demonstratedsignificant biological heparin activity.

In this example, implantable medical devices in the form of endoluminalprostheses were heparinized using a process substantially equivalent toExample 3. Aldehyde modified polyethylene glycol (1,000 MW) was attachedto the coating layer via a labile covalent bond in a mannersubstantially equivalent to that described in Example 12 excluding theaddition of NaCNBH₃. It is important to note that the reducing agent,NaCNBH₃, was not added during the conjugation of aldehyde modifiedpolyethylene glycol. The bond formed between primary amines andaldehydes, when left in the un-reduced state, is labile. The sampleswere then rinsed in DI water for fifteen minutes (15 min), borate buffersolution (10.6 g boric acid, 2.7 g NaOH and 0.7 g NaCl dissolved in 1 LDI water, pH 9.0) for twenty minutes (20 min), and finally in DI waterfor fifteen minutes (15 min) followed by lyophilization of the entireconstruction to produce dry heparin and polyethylene glycol bound to theePTFE material. Dexamethasone was then non-covalently admixed with thiscomposition as described in Example 2. The heparinized endoluminalprostheses were then mechanically compacted, mechanically expanded, cutfor testing, rinsed, and assayed for ATIII binding in a mannersubstantially equivalent to that described in Example 5.

The sample prepared as described in this example had a meananti-thrombin III binding activity of 188 pmol/cm² (n=3).

Example 33

This example describes the synthesis and covalent attachment ofbiologically active dermatan disulfate (17) to a covering material, orcoating layer, placed on an expanded polytetrafluoroethylene (ePTFE)material with HCII binding activity.

In accordance with U.S. Pat. No. 5,922,690, which is incorporated hereinby reference, dermatan disulfate is produced. This material is furtherprocessed in accordance with U.S. Pat. No. 6,653,457, which isincorporated herein by reference, to produce an aldehyde modifieddermatan disulfate composition made according to U.S. Pat. No.4,613,665, which is incorporated herein by reference, for attachment toa covering material, or coating layer, placed on an expandedpolytetrafluoroethylene (ePTFE) material.

An ePTFE material in sheet form is obtained from W.L. Gore & Associates,Inc., Flagstaff, Ariz. under the tradename GORE™ Microfiltration Media(GMM-406). A covering material in the form of a base coating is appliedto the ePTFE material by mounting the material on a ten centimeter (10cm) diameter plastic embroidery hoop and immersing the supported ePTFEmaterial first in 100% isopropyl alcohol (IPA) for about five minutes (5min) and then in a solution of LUPASOL® polyethylene imine (PEI) and IPAin a one to one ratio (1:1). LUPASOL® water-free PEI is obtained fromBASF and diluted to a concentration of about four percent (4%) andadjusted to pH 9.6. Following immersion of the ePTFE material in thesolution for about fifteen minutes (15 min), the material is removedfrom the solution and rinsed in deionized (DI) water at pH 9.6 forfifteen minutes (15 min). PEI remaining on the ePTFE material iscross-linked with a 0.05% aqueous solution of glutaraldehyde (obtainedfrom Amresco) at pH 9.6 for fifteen minutes (15 min). Additional PEI isadded to the construction by placing the construction in a 0.5% aqueoussolution of PEI at pH 9.6 for fifteen minutes (15 min) and rinsing againin DI water at pH 9.6 for fifteen minutes (15 min). The imine formed asa result of the reaction between glutaraldehyde and the PEI layer isreduced with a sodium cyanborohydride (NaCNBH₃) solution (5 g dissolvedin 1 L DI water, pH 9.6) for fifteen minutes (15 min) and rinsed in DIwater for thirty minutes (30 min).

An additional layer of PEI is added to the construction by immersing theconstruction in 0.05% aqueous glutaraldehyde solution at pH 9.6 forfifteen minutes (15 min), followed by immersion in a 0.5% aqueoussolution of PEI at pH 9.6 for fifteen minutes (15 min). The constructionis then rinsed in DI water at pH 9.6 for fifteen minutes (15 min). Theresultant imines are reduced by immersing the construction in a solutionof NaCNBH₃ (5 g dissolved in 1 L DI water, pH 9.6) for fifteen minutes(15 min) followed by a rinse in DI water for thirty minutes (30 min). Athird layer is applied to the construction by repeating these steps. Theresult is a porous hydrophobic fluoropolymeric base material having ahydrophilic cross-linked polymer base coat on substantially all of theexposed and interstitial surfaces of the base material.

An intermediate chemical layer is attached to the polymer base coat inpreparation for placement of another layer of PEI on the construction.The intermediate ionic charge layer is made by incubating theconstruction in a solution of dextran sulfate (Amersham PharmaciaBiotech) and sodium chloride (0.15 g dextran sulfate and 100 g NaCldissolved in 1 L DI water, pH 3) at 60° C. for ninety minutes (90 min)followed by rinsing in DI water for fifteen minutes (15 min).

A layer of PEI, referred to herein as a “capping layer” is attached tothe intermediate layer by placing the construction in a 0.3% aqueoussolution of PEI (pH 9) for about forty-five minutes (45 min) followed bya rinse in a sodium chloride solution (50 g NaCl dissolved in 1 L DIwater) for twenty minutes (20 min). A final DI water rinse is conductedfor twenty minutes (20 min).

Aldehyde modified dermatan disulfate is attached, or conjugated, to thePEI layer(s) by placing the construction in a dermatandisulfate-containing sodium chloride salt solution (1.5 g dermatandisulfate, 29.3 g NaCl dissolved in 1 L DI water, pH 3.9) for onehundred twenty minutes (120 min) at sixty degrees centigrade (60° C.). A2.86 mL volume of a 2.5% (w/v) aqueous NaCNBH₃ solution is added to theone liter (1 L) dermatan disulfate solution prior to adding the samples.The samples are then rinsed in DI water for fifteen minutes (15 min),borate buffer solution (10.6 g boric acid, 2.7 g NaOH and 0.7 g NaCldissolved in 1 L DI water, pH 9.0) for twenty minutes (20 min), andfinally in DI water for fifteen minutes (15 min) followed bylyophilization of the entire construction to produce dry dermatandisulfate bound to the ePTFE material. The presence and uniformity ofthe dermatan disulfate is determined by staining samples of theconstruction on both sides with toluidine blue. The staining produces anevenly purpled surface indicating dermatan disulfate is present anduniformly bound to the ePTFE material.

Samples approximately one square centimeter (1 cm²) in size are cut fromthe construction and assayed for dermatan disulfate activity bymeasuring the capacity of the end point attached dermatan disulfate tobind HCII. Calculations of dermatan disulfate activity on surfaces inthe present invention were conducted using the surface area of only oneside of the sample material, although the entire sample, includinginterstices, may have dermatan disulfate immobilized thereon. Thedermatan disulfate activity is assayed by measuring the ability, orcapacity, of the end-point attached dermatan disulfate to bind a knownquantity of heparin cofactor II (HC II). The results are expressed aspicomoles heparin cofactor II (HC II) bound per square centimeter ofsubstrate material (pmol HC II/cm² substrate material). Samplesapproximately one square centimeter (1 cm²) in size are cut from theconstruction and assayed for dermatan disulfate activity by measuringthe capacity of the end point attached dermatan disulfate to bindheparin cofactor II (HCII). The measurement of dermatan disulfateactivity is similar to that described previously for heparin activity byLarsen M. L., et al., in “Assay of plasma heparin using thrombin and thechromogenic substrate H-D-Phe-Pip-Arg-pNA (S-2238).” Thromb Res13:285-288 (1978) and Pasche B., et al., in “A binding of antithrombinto immobilized heparin under varying flow conditions.” Artif. Organs15:281-491 (1991). For the dermatan disulfate activity assay, HCII isallowed to bind to the dermatan disulfate surface, eluted from thesurface by an excess of soluble dermatan disulfate, and combined withthrombin in a colorimetric assay for thrombin activity. The assayindirectly determines the amount of HCII present by measuringHCII-mediated inhibition of human thrombin. The amount of HCII isdetermined from a standard curve derived by mixing known amounts ofdermatan disulfate, HCII, thrombin, and a synthetic thrombin substrate(known as an amidolytic assay). A similar approach for measuring solubledermatan sulfate activity has been previously described by Dupouy D., etal., in “A simple method to measure dermatan sulfate at sub-microgramconcentrations in plasma.” Thromb. Haemost. 60:236-239 (1988). Theresults are expressed as amount of HCII bound per unit surface areasubstrate material in picomoles per square centimeter (pmol/cm2). Allsamples are maintained in a wet condition throughout the assay. It isimportant to note that while the approximately one square centimeter (1cm²) samples each have a total surface area of two square centimeters (2cm²) if both sides of the material are considered, only one surface onthe sample (i.e., 1 cm²) is used for calculating HCII-dermatandisulfate-binding activity in pmol/cm².

In an alternative method, dermatan disulfate activity is directlyquantified by measuring the amount of radiolabeled HCII bound to thedermatan disulfate-immobilized construct. This technique is similar tomethods described for measuring antithrombin III binding to immobilizedheparin constructs by Du Y. J., et al., in “Protein adsorption onpolyurethane catheters modified with a novel antithrombin-heparincovalent complex.” J. Biomed. Mater. Res. 80A:216-225 (2007). Thedermatan disulfate construct is incubated with a solution of HCII thathas been covalently labeled with the radioisotope Iodine-125 (¹²⁵I).After incubation the surface is repeatedly rinsed and the amount ofradiation emitted from the construct is measured by a gamma counter.Because the ratio of emission to HCII mass is known, the amount of HCIIcan be determined. The results are expressed as amount of HCII bound perunit surface area substrate material in picomoles per square centimeter(pmol/cm²).

HC II binding activity per surface area of substrate material is definedas the number of picomoles of HC II bound per apparent surface area ofcovered or uncovered substrate material. The apparent substrate surfacearea does not take into account multiple covered surfaces nor porosityconsiderations of a porous substrate material. If the substrate materialis porous, the effect of porosity on surface area is not considered forthese calculations. For example, the apparent surface area of acylindrical tubular ePTFE vascular graft (which is made of a porousmaterial) with end-point attached heparin immobilized on substratematerial comprising the inner surface of the tubular graft is calculatedas it is for any cylindrical geometry as 2πrL: where r is the graftinner radius; L is the axial length; and π is the number pi. It isimportant to note that the porous nature of ePTFE and its effect onsurface area is not accounted for herein. Accordingly, non-poroussubstrate materials that are cut into squares for analysis are taken tohave a surface area of the length multiplied by the width. The resultsare expressed as amount of HCII bound per unit surface area substratematerial in picomoles per square centimeter (pmol/cm²). All samples aremaintained in a wet condition throughout the assay. It is important tonote that while the approximately one square centimeter (1 cm²) sampleseach have a total surface area of two square centimeters (2 cm²) if bothsides of the material are considered, only one surface on the sample(i.e., 1 cm²) is used for calculating HCII binding activity in pmol/cm².

Some samples prepared as described in this example have a heparincofactor II binding activity of greater than 5 pmol/cm². Other sampleshave a heparin cofactor II binding activity of greater than 12 pmol/cm².Yet other samples have a heparin cofactor II binding activity of greaterthan 20 pmol/cm². These results demonstrate the ability to produce asurface with HC II binding activity.

Example 34

This example describes the covalent attachment of biologically activedermatan disulfate (17) to a covering material, or coating layer, placedon an expanded polytetrafluoroethylene (ePTFE) material followed by theadmixture of an additional biologically compatible organic composition.The first biologically compatible organic composition is USP heparin, apolysaccharide, pharmaceutical, hydrophilic molecule, andantithrombogenic compound. The second compound is polyethylene glycol, asynthetic and hydrophilic compound. This construct demonstrates greaterthan 5 pmol/cm² of HCII binding activity.

A construct is prepared as described in Example 33. Following thecovalent attachment of dermatan disulfate, these constructions areexposed to the following biologically compatible organic compositions:USP grade heparin sodium (Celsus) and polyethylene glycol (20,000molecular weight, Sigma) at concentrations of 0.5 g per 100 ml DI wateradjusted to pH 9.6. Each of these solutions is referred to herein as a“treatment solution.” To expose a particular dermatandisulfate-containing construction to a particular treatment solution,the construction is placed into a two liter (2 L) beaker and one hundredmilliliters (100 ml) of treatment solution is added, sufficient tocompletely immerse the construction in the treatment solution. Eachconstruction is exposed to the treatment solution for one hour (1 hr) atsixty degrees centigrade (60° C.). The construction is removed from thesolution and lyophilized.

Next, each construction (including controls) is washed in DI water forfifteen minutes (15 min), borate buffer solution (10.6 g boric acid, 2.7g NaOH and 0.7 g NaCl dissolved in 1 L DI water, pH 9.0) for twentyminutes (20 min), and finally a rinse in DI water for fifteen minutes(15 min).

The samples prepared as described in this example have a heparincofactor II binding activity of greater than 5 pmol/cm² when tested asdescribed in Example 33. These results demonstrate the ability toproduce a surface with HC II binding activity with the additionaladmixed molecules.

Example 35

This example describes the covalent attachment of biologically activedermatan disulfate (17) to a covering material, or coating layer, placedon an expanded polytetrafluoroethylene (ePTFE) material followed by theadmixture of an additional biologically compatible organic composition.This compound, dexamethasone, is a synthetic and hydrophobicpharmaceutical.

The above-described construction coated with a covering material isexposed to a dexamethasone solution containing 0.5 g per 100 ml ethanolwith no pH adjustment. This solution is referred to herein as a“treatment solution.” To expose a particular heparin-containingconstruction to this treatment solution, the construction is placed intoa two liter (2 L) beaker and one hundred milliliters (100 ml) oftreatment solution is added, sufficient to completely immerse theconstruction in the treatment solution. This construction is exposed tothe treatment solution for one hour (1 hr) at sixty degrees centigrade(60° C.). The construction is then removed from the solution andlyophilized.

Next, each construction (including controls) is washed in DI water forfifteen minutes (15 min), borate buffer solution (10.6 g boric acid, 2.7g NaOH and 0.7 g NaCl dissolved in 1 L DI water, pH 9.0) for twentyminutes (20 min), and finally a rinse in DI water for fifteen minutes(15 min).

The samples prepared as described in this example have a heparincofactor II binding activity of greater than 5 pmol/cm² when tested asdescribed in Example 33. These results demonstrate the ability toproduce a surface with HC II binding activity with an additional admixedmolecule.

Example 36

This example describes the covalent attachment of biologically activedermatan disulfate (17) to a covering material, or coating layer, placedon an expanded polytetrafluoroethylene (ePTFE) material followed by theadmixture of biologically compatible organic compositions. Theconstruction exhibits significant HCII binding after exposure toethylene oxide (EtO) sterilization.

Samples with biologically active dermatan disulfate are prepared asdescribed in Example 33. The biologically compatible organiccompositions polyethylene glycol, USP heparin, and dexamethasone areadmixed with separate samples as described in Examples 34 and 35.

In preparation for EtO sterilization, each lyophilized construction isplaced and sealed in a Tower DUALPEEL® Self-Seal Pouch (AllegianceHealthcare Corp., McGaw Park, Ill.). Ethylene oxide sterilization iscarried out under conditions of conditioning for one hour (1 hr), an EtOgas dwell time of one hour (1 hr), a set point temperature of fifty-fivedegree centigrade (55° C.), and an aeration time of twelve hours (12hr).

After EtO sterilization, each construction (including controls) isremoved from its pouch and washed in DI water for fifteen minutes (15min), borate buffer solution (10.6 g boric acid, 2.7 g NaOH and 0.7 gNaCl dissolved in 1 L DI water, pH 9.0) for twenty minutes (20 min), andfinally a rinse in DI water for fifteen minutes (15 min).

The samples prepared as described in this example have a heparincofactor II binding activity of greater than 5 pmol/cm² when tested asdescribed in Example 33. These results demonstrate the ability toproduce a surface with HC II binding activity, with additional admixedmolecules, following EtO sterilization

Example 37

This example describes the construction of an embodiment of the presentinvention in which dermatan disulfate (17) HCII binding on a medicaldevice substrate is greater than 5 pmol/cm² following mechanicalcompaction and expansion.

The implantable medical device used in this example is in the form of anitinol wire reinforced tube made of a porous, expanded,polytetrafluoroethylene (ePTFE) material obtained from W.L. Gore &Associates, Inc., Flagstaff, Ariz. under the tradename VIABAHN®Endoprosthesis. The tubular device is fifteen centimeters (15 cm) inlength and six millimeters (6 mm) in diameter.

The VIABAHN® Endoprosthesis is constrained within a delivery catheterand required removal from the catheter before immobilizing heparinthereon. Each catheter-constrained device is removed for processing bypulling a release cord attached to a constraining sheath and releasingthe sheath from around the device. Once unconstrained, each device isexpanded and used as a separate substrate material. Each substratematerial (endoprosthetic device) is immersed in a PEI solution (5% in DIwater) and IPA (USP grade) in a volume percent ratio of 30:70,respectively, for about twelve hours (12 hr) to place a polymericcovering material (18) on the substrate material (12). The polymericcovering material (18) has a multiplicity of reactive chemical groups(16) to which a plurality of aldehyde-modified dermatan disulfatemolecules (17) are eventually end point attached.

At least one additional layer of covering material (18 a, 18 b) isplaced on the first PEI layer (18). This is performed by placing eachendoprosthetic device within a separate silicone tube and the tubeconnected to a peristaltic pump and solution-reservoir. This allows anadditional solution containing a covering material to be repeatedlypassed through the center of the tubular medical device to coatprimarily the inside surfaces of the device.

With each endoprosthesis contained within one of these dynamic flowsystems, a covering material (18) in the form of an aqueous solution of0.10% (pH 9.0) PEI and IPA in a volume percent ratio of 45:55,respectively, is passed through the device for about twenty minutes (20min). Each device is then rinsed in DI water (pH 9.0) for five minutes(5 min) and the PEI layers cross-linked (19) by exposure to a 0.05%aqueous glutaraldehyde solution (pH 9.0) for twenty minutes (20 min).The devices are then rinsed again with an aqueous solution of PEI(0.10%, pH 9.0) for five minutes (5 min). The resultant imines arereduced with a sodium cyanborohydride solution (5 g in 1 L DI water, pH9.0) for fifteen minutes (15 min) and rinsed in DI water for thirtyminutes (30 min).

An intermediate ionic charge layer is placed on the cross-linked PEIlayer(s) of each device by flowing a solution of dextran sulfate (0.15 gdextran sulfate and one hundred grams sodium chloride (100 g NaCl)dissolved in one liter (1 L) of DI water, pH 3) through the dynamic flowsystem and over the PEI layer at sixty degrees centigrade (60° C.) forabout ninety minutes (90 min). This is followed by rinsing the systemwith DI water for fifteen minutes (15 min).

A “capping” layer (18 b) of PEI is added to the ionically chargeddextran sulfate layer (18 a) by flowing an aqueous solution of PEI(0.075%, pH 9.0) through the dynamic flow system for about forty-fiveminutes (45 min) followed by a rinse in a sodium chloride solution (50 gNaCl dissolved in 1 L DI water) for fifteen minutes (15 min). The rinseis followed by a brief DI water flush for about two and a half minutes(2.5 min).

Aldehyde modified dermatan disulfate is attached, or conjugated, to thePEI layer(s) by placing the construction in a dermatandisulfate-containing sodium chloride salt solution (1.5 g dermatandisulfate, 29.3 g NaCl dissolved in 1 L DI water, pH 3.9) for onehundred twenty minutes (120 min) at sixty degrees centigrade (60° C.). A2.86 mL volume of a 2.5% (w/v) aqueous NaCNBH₃ solution is added to theone liter (1 L) dermatan disulfate solution prior to adding the samples.The samples are then rinsed in DI water for fifteen minutes (15 min),borate buffer solution (10.6 g boric acid, 2.7 g NaOH and 0.7 g NaCldissolved in 1 L DI water, pH 9.0) for twenty minutes (20 min), andfinally in DI water for fifteen minutes (15 min) followed bylyophilization of the entire construction to produce dry dermatandisulfate bound to the ePTFE material. The presence and uniformity ofthe dermatan disulfate is determined by staining samples of theconstruction on both sides with toluidine blue. The staining produces anevenly purpled surface indicating dermatan disulfate is present anduniformly bound to the ePTFE material.

To compress and compact the endoluminal devices on a delivery system,each endoprosthesis is pulled through a tapered funnel with a fixeddiameter. Each endoprosthesis has six (6) sutures (GORE-TEX® CV-0, 0N05)sewn through one end to pull the devices through the funnel. Each deviceis pulled through the opening of a twenty-five milliliter (25 ml) pipettip (Falcon®, product #357525) with a diameter of about threemillimeters (3 mm) and into a glass tube with a diameter of about 3.1 mmto hold it in the compacted state.

After compaction, each endoprosthesis is deployed in a 0.9% aqueoussaline solution at thirty-seven degree centigrade (37° C.). Eachendoprosthesis is prepared for testing by washing in DI water forfifteen minutes (15 min), followed by a rinse in borate buffer solution(10.6 g boric acid 2.7 g NaOH, 0.7 g NaCl, dissolved in 1 L of DI water,pH 9.0) for twenty minutes (20 min) and a final fifteen minute (15 min)DI water rinse.

Samples of dermatan disulfate-containing material from eachendoprosthesis (approx. 0.5 cm long) are cut and the bound heparinmeasured for biological activity using the above-described heparincofactor II (HCII) binding assay (Example 33). Samples are kept wetthroughout the assay process. The results are expressed as heparincofactor II binding per unit of substrate surface area (pmol/cm²substrate material).

The samples in this example have a heparin cofactor II binding activitygreater than 5 pmol/cm². These results demonstrate the ability toproduce a surface with HCII binding activity following mechanicalcompaction and expansion.

Example 38

This example describes the covalent attachment of biologically activedermatan disulfate (17) to a covering material, or coating layer, placedon an expanded polytetrafluoroethylene (ePTFE) medical device followedby the admixture of biologically compatible organic compositions. Theconstruction exhibits significant HCII binding after exposure tomechanical compaction and expansion.

Samples with biologically active dermatan disulfate are prepared asdescribed in Example 37. The biologically compatible organiccompositions polyethylene glycol, USP heparin, and dexamethasone areadmixed with separate samples as described in Examples 34 and 35.

The constructions are further mechanically compacted, deployed, rinsed,sampled, and tested as described in Example 37. Constructions treated inthis way exhibit greater than 5 pmol/cm² HCII binding activity whentested as described in Example 33.

Example 39

This example describes the covalent attachment of biologically activedermatan disulfate (17) and heparin (17), to a covering material, orcoating layer, placed on an expanded polytetrafluoroethylene (ePTFE)material with HCII binding greater than 5 pmol/cm² and antithrombin III(ATIII) binding of greater than 5 pmol/cm².

In accordance with U.S. Pat. No. 6,653,457, which is incorporated hereinby reference, an aldehyde modified heparin composition is made accordingto U.S. Pat. No. 4,613,665, which is incorporated herein by reference.

An ePTFE construction is prepared as described in Example 33, with theexception of the procedure related to the attachment of aldehydemodified dermatan disulfate. In this treatment step, instead of only 1.5g of dermatan disulfate, 1.5 g of aldehyde modified heparin and 1.5 g ofaldehyde modified dermatan disulfate are added to the solution.

HCII binding activity is measured as described in Example 33. Samplesare also assayed for ATII activity by measuring the capacity of the endpoint attached heparin to bind ATIII. The assay is described by LarsenM. L., et al., in “Assay of plasma heparin using thrombin and thechromogenic substrate H-D-Phe-Pip-Arg-pNA (S-2238).” Thromb Res13:285-288 (1978) and Pasche B., et al., in “A binding of antithrombinto immobilized heparin under varying flow conditions.” Artif. Organs15:281-491 (1991). The results are expressed as amount of ATIII boundper unit surface area substrate material in picomoles per squarecentimeter (pmol/cm²). All samples are maintained in a wet conditionthroughout the assay. It is important to note that while theapproximately one square centimeter (1 cm²) samples each have a totalsurface area of two square centimeters (2 cm²) if both sides of thematerial are considered, only one surface on the sample (i.e., 1 cm²) isused for calculating ATIII heparin-binding activity in pmol/cm².

Constructions prepared and tested as described in this example exhibitboth greater than 5 pmol/cm² HCII binding activity and an ATIII bindingof greater than 5 pmol/cm².

Example 40

This example describes the covalent attachment of biologically activedermatan disulfate (17) and heparin (17), to a covering material, orcoating layer, placed on an expanded polytetrafluoroethylene (ePTFE)material with the inclusion of an admixed biologically compatibleorganic composition and resultant HCII binding greater than 5 pmol/cm²and ATIII binding of greater than 5 pmol/cm² following EtOsterilization.

Samples are prepared as described in Example 39. The biologicallycompatible organic compositions polyethylene glycol, USP heparin, anddexamethasone are admixed with separate samples as described in Examples34 and 35. These samples are further sterilized as described in Example36.

Constructions prepared and tested as described in this example exhibitboth greater than 5 pmol/cm² HCII binding activity and an ATIII bindingof greater than 5 pmol/cm².

Example 41

This example describes the covalent attachment of biologically activedermatan disulfate (17) and heparin (17), to a covering material, orcoating layer, placed on an expanded polytetrafluoroethylene (ePTFE)with the inclusion of an admixed biologically compatible organiccomposition (11) and resultant HCII binding greater than 5 pmol/cm² andATIII binding of greater than 5 pmol/cm² following mechanical compactionand expansion.

VIABAHN® Endoprostheses are prepared as described in Example 37, withthe exception of the procedure related to the attachment of aldehydemodified dermatan disulfate. In this treatment step, instead of only 1.5g of dermatan disulfate, 1.5 g of aldehyde modified heparin and 1.5 g ofaldehyde modified dermatan disulfate are added to the solution. Next,the biologically compatible organic compositions polyethylene glycol,USP heparin, and dexamethasone are admixed with separate samples asdescribed in Examples 34 and 35. Prior to testing, the samples are thenrinsed as described in Example 34.

Constructions prepared and tested as described in this example exhibitboth greater than 5 pmol/cm² HCII binding activity and an ATIII bindingof greater than 5 pmol/cm² following mechanical compaction andexpansion.

Example 42

This example describes the covalent attachment of biologically activedermatan disulfate (17) to a covering material, or coating layer, placedon an expanded polytetrafluoroethylene (ePTFE) material followed bysterilization. The construction exhibits significant HCII binding afterexposure to ethylene oxide (EtO) sterilization.

Samples with biologically active dermatan disulfate are prepared asdescribed in Example 33 and sterilized as described in Example 36.Constructions prepared and tested as described in this example exhibitgreater than 5 pmol/cm² HCII binding activity.

Example 43

This example describes the covalent attachment of biologically activedermatan disulfate (17) and heparin (17), to a covering material, orcoating layer, placed on an expanded polytetrafluoroethylene (ePTFE)material with HCII binding greater than 5 pmol/cm² and antithrombin III(ATIII) binding of greater than 5 pmol/cm² following EtO sterilization.

Samples are prepared as described in example 39, subjected tosterilization as described in Example 36, and tested as described inExample 39. Samples prepared and tested as described in this exampleexhibit greater than 5 pmol/cm² HCII binding activity and an ATIIIbinding of greater than 5 pmol/cm².

Example 44

This example describes the construction of an embodiment of the presentinvention in which dermatan disulfate (17) HCII binding on a medicaldevice substrate is greater than 5 pmol/cm² following sterilization.

Samples with biologically active dermatan disulfate are prepared asdescribed in Example 37, sterilized as described in Example 36, andtested as described in Example 33. Samples prepared and tested asdescribed in this example exhibit greater than 5 pmol/cm² HCII bindingactivity.

The invention claimed is:
 1. A medical device comprising: a polymericsubstrate material, a plurality of biologically active entities havingheparin cofactor II binding activity covalently attached to at least aportion of said polymeric substrate material; and a biologicallycompatible composition combined with said plurality of biologicallyactive entities, wherein said biologically active entities have aheparin cofactor II binding activity of at least 5 picomoles heparincofactor II per square centimeter (pmol/cm²).
 2. The medical device ofclaim 1 wherein said biologically compatible composition is covalentlycombined with said plurality of biologically active entities.
 3. Themedical device of claim 1 wherein said biologically compatiblecomposition is non-covalently combined with said plurality ofbiologically active entities.
 4. The medical device of claim 1 whereinsaid biologically compatible composition is covalently combined withsaid polymeric substrate material.
 5. The medical device of claim 1wherein said biologically compatible composition is non-covalentlycombined with said polymeric substrate material.
 6. The medical deviceof claim 1 wherein said plurality of biologically active entitiescomprises a glycosaminoglycan.
 7. The medical device of claim 1 whereinsaid plurality of biologically active entities comprises dermatandisulfate.
 8. The medical device of claim 1 wherein said plurality ofbiologically active entities comprises end-point attached dermatandisulfate.
 9. The medical device of claim 1 wherein said medical deviceis capable of releasing at least a portion of said biologicallycompatible composition, and wherein upon exposure to a 0.15M phosphatebuffer solution having a temperature of about thirty-seven degreescentigrade and a substantially neutral pH, at least a portion of saidbiologically compatible composition is released from said medicaldevice.
 10. The medical device of claim 1 wherein said biologicallycompatible composition comprises an organic compound.
 11. The medicaldevice of claim 10 wherein said organic compound is a polysaccharide.12. The medical device of claim 11 wherein said polysaccharide is aglycosaminoglycan.
 13. The medical device of claim 11 wherein saidpolysaccharide is dextran.
 14. The medical device of claim 11 whereinsaid polysaccharide is dextran sulfate.
 15. The medical device of claim1 wherein said biologically compatible composition is polyethyleneglycol.
 16. The medical device of claim 1 wherein said biologicallycompatible composition is an antiproliferative agent.
 17. The medicaldevice of claim 16 wherein said antiproliferative agent isdexamethasone.
 18. The medical device of claim 1 wherein saidbiologically compatible composition comprises a synthetic non-polarmolecule.
 19. The medical device of claim 1 wherein said biologicallycompatible composition comprises an inorganic compound.
 20. The medicaldevice of claim 19 wherein said inorganic compound comprises aphosphate.
 21. The medical device of claim 1 wherein said medical deviceis capable of being sterilized and said plurality of biologically activeentities have a heparin cofactor II binding activity of at least 5picomoles heparin cofactor II per square centimeter (pmol/cm²) substratematerial when measured following said sterilization of said polymericsubstrate material.
 22. The medical device of claim 1 wherein saidmedical device is capable of being compacted and expanded and saidplurality of biologically active entities have a heparin cofactor IIbinding activity of at least 5 picomoles heparin cofactor II per squarecentimeter (pmol/cm²) substrate material when measured following saidcompaction and expansion of said polymeric substrate material.
 23. Themedical device of claim 1 wherein said biologically compatiblecomposition comprises the co-immobilization of heparin and dermatandisulfate.
 24. A medical device comprising: a polymeric substratematerial, a first plurality of biologically active entities havingheparin cofactor II binding activity and a second plurality ofbiologically active entities having anti-thrombin Ill binding activitycovalently attached to at least a portion of said polymeric substratematerial; and a biologically compatible composition combined with saidfirst plurality of biologically active entities and said secondplurality of biologically active entities, wherein said biologicallyactive entities have a heparin cofactor II binding activity of at least5 picomoles heparin cofactor II per square centimeter (pmol/cm²) and atleast 5 picomoles anti-thrombin Ill per square centimeter.
 25. Themedical device of claim 24 wherein said biologically compatiblecomposition is covalently combined with said said first plurality ofbiologically active entities and said second plurality of biologicallyactive entities.
 26. The medical device of claim 24 wherein saidbiologically compatible composition is non-covalently combined with saidsaid first plurality of biologically active entities and said secondplurality of biologically active entities.
 27. The medical device ofclaim 24 wherein said biologically compatible composition is covalentlycombined with said polymeric substrate material.
 28. The medical deviceof claim 24 wherein said biologically compatible composition isnon-covalently combined with said polymeric substrate material.
 29. Themedical device of claim 24 wherein said first plurality of biologicallyactive entities comprises a glycosaminoglycan.
 30. The medical device ofclaim 24 wherein said first plurality of biologically active entitiescomprises dermatan disulfate.
 31. The medical device of claim 24 whereinsaid first plurality of biologically active entities comprises end-pointattached dermatan disulfate.
 32. The medical device of claim 24 whereinat least a portion of said biologically compatible composition isreleased from said medical device in a 0.15M phosphate buffer solutionhaving a temperature of about thirty-seven degrees centigrade and asubstantially neutral pH.
 33. The medical device of claim 24 whereinsaid biologically compatible composition comprises an organic compound.34. The medical device of claim 33 wherein said organic compound is apolysaccharide.
 35. The medical device of claim 34 wherein saidpolysaccharide is a glycosaminoglycan.
 36. The medical device of claim34 wherein said polysaccharide is dextran.
 37. The medical device ofclaim 34 wherein said polysaccharide is dextran sulfate.
 38. The medicaldevice of claim 24 wherein said biologically compatible composition ispolyethylene glycol.
 39. The medical device of claim 24 wherein saidbiologically compatible composition is an antiproliferative agent. 40.The medical device of claim 39 wherein said antiproliferative agent isdexamethasone.
 41. The medical device of claim 24 wherein saidbiologically compatible composition comprises a synthetic non-polarmolecule.
 42. The medical device of claim 24 wherein said biologicallycompatible composition comprises an inorganic compound.
 43. The medicaldevice of claim 42 wherein said inorganic compound comprises aphosphate.
 44. The medical device of claim 24 wherein said firstplurality of biologically active entities have a heparin cofactor IIbinding activity of at least 5 picomoles heparin cofactor II per squarecentimeter (pmol/cm²) substrate material following sterilization of saidpolymeric substrate material.
 45. The medical device of claim 24 whereinsaid first plurality of biologically active entities have a heparincofactor II binding activity of at least 5 picomoles heparin cofactor IIper square centimeter (pmol/cm²) substrate material following compactionand expansion of said polymeric substrate material.
 46. The medicaldevice of claim 24 wherein said biologically compatible compositioncomprises the co-immobilization of heparin and dermatan disulfate.